Donald S. Kirkpatrick

Advances in biological mass spectrometry have resulted in the development of numerous strategies for the large-scale quantification of protein expression levels within cells. These measurements of protein expression are most commonly accomplished through differential incorporation of stable isotopes into cellular proteins. Several variations of the stable isotope quantification method have been demonstrated, differing in isotope composition and incorporation strategy. In general, the majority of these methods establish only relative quantification of expressed proteins. To address this, the absolute quantification (AQUA) strategy was developed for the precise determination of protein expression and post-translational modification levels. The AQUA method relies on the use of a synthetic internal standard peptide that is introduced at a known concentration to cell lysates during digestion. This AQUA peptide precisely mimics a peptide produced during proteolysis of the target protein, except that it is enriched in certain stable isotopes. Analysis of the proteolyzed sample by a selected reaction monitoring (SRM) experiment in a tandem mass spectrometer results in the direct detection and quantification of both the native peptide and isotope labeled AQUA internal standard peptide. As an example, the development and application of a method to measure a tryptic peptide representing the amount of polyubiquitin chain formation through lysine 48 (K48) is presented. The simplicity and sensitivity of the method, coupled with the widespread availability of tandem mass spectrometers, make the AQUA strategy a highly useful procedure for measuring the levels of proteins and post-translational modifications directly from cell lysates.

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.

Ubiquitination of the EGF receptor (EGFR) is believed to play a critical role in regulating both its localization and its stability. To elucidate the role of EGFR ubiquitination, tandem mass spectrometry was used to identify six distinct lysine residues within the kinase domain of the EGFR, which can be conjugated to ubiquitin following growth factor stimulation. Substitution of these lysine residues with arginines resulted in a dramatic decrease in overall ubiquitination but preserved normal tyrosine phosphorylation of EGFR. Ubiquitination-deficient EGFR mutants displayed a severe defect in their turnover rates but were internalized at rates comparable to those of wild-type receptors. Finally, quantitative mass spectrometry demonstrated that more than 50% of all EGFR bound ubiquitin was in the form of polyubiquitin chains, primarily linked through Lys63. Taken together, these data provide direct evidence for the role of EGFR ubiquitination in receptor targeting to the lysosome and implicate Lys63-linked polyubiquitin chains in this sorting process.

Although ubiquitin-enriched protein inclusions represent an almost invariant feature of neurodegenerative diseases, the mechanism underlying their biogenesis remains unclear. In particular, whether the topology of ubiquitin linkages influences the dynamics of inclusions is not well explored. Here, we report that lysine 48 (K48)- and lysine 63 (K63)-linked polyubiquitination, as well as monoubiquitin modification contribute to the biogenesis of inclusions. K63-linked polyubiquitin is the most consistent enhancer of inclusions formation. Under basal conditions, ectopic expression of K63 mutant ubiquitin in cultured cells promotes the accumulation of proteins and the formation of intracellular inclusions in the apparent absence of proteasome impairment. When co-expressed with disease-associated tau and SOD1 mutants, K63 ubiquitin mutant facilitates the formation of tau- and SOD-1-positive inclusions. Moreover, K63-linked ubiquitination was found to selectively facilitate the clearance of inclusions via autophagy. These data indicate that K63-linked ubiquitin chains may represent a common denominator underlying inclusions biogenesis, as well as a general cellular strategy for defining cargo destined for the autophagic system. Collectively, our results provide a novel mechanistic route that underlies the life cycle of an inclusion body. Harnessing this pathway may offer innovative approaches in the treatment of neurodegenerative disorders.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Ubiquitin chains serve as a recognition motif for the proteasome, a multisubunit protease, which degrades its substrates into polypeptides while releasing ubiquitin for reuse. Yeast proteasomes contain two deubiquitinating enzymes, Ubp6 and Rpn11. Rpn11 promotes protein breakdown through its degradation-coupled activity. In contrast, we show here that Ubp6 has the capacity to delay the degradation of ubiquitinated proteins by the proteasome. However, delay of degradation by Ubp6 does not require its catalytic activity, indicating that Ubp6 has both deubiquitinating activity and proteasome-inhibitory activity. Delay of degradation by Ubp6 appears to provide a time window allowing gradual deubiquitination of the substrate by Ubp6. Rpn11 catalyzes en bloc chain removal, and Ubp6 interferes with degradation at or upstream of this step, so that degradation delay by Ubp6 is accompanied by a switch in the mode of ubiquitin chain processing. We propose that Ubp6 regulates both the nature and magnitude of proteasome activity.

LRRK2 autophosphorylation on Ser 1292 may be a useful indicator of kinase activity, providing a readout for screening candidate LRRK2 inhibitors.

Article26 November 2010free access c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling Jasmin N Dynek Jasmin N Dynek Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Tatiana Goncharov Tatiana Goncharov Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Erin C Dueber Erin C Dueber Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Anna V Fedorova Anna V Fedorova Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Anita Izrael-Tomasevic Anita Izrael-Tomasevic Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Lilian Phu Lilian Phu Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Elizabeth Helgason Elizabeth Helgason Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Wayne J Fairbrother Wayne J Fairbrother Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Kurt Deshayes Kurt Deshayes Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Donald S Kirkpatrick Donald S Kirkpatrick Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Domagoj Vucic Corresponding Author Domagoj Vucic Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Jasmin N Dynek Jasmin N Dynek Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Tatiana Goncharov Tatiana Goncharov Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Erin C Dueber Erin C Dueber Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Anna V Fedorova Anna V Fedorova Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Anita Izrael-Tomasevic Anita Izrael-Tomasevic Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Lilian Phu Lilian Phu Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Elizabeth Helgason Elizabeth Helgason Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Wayne J Fairbrother Wayne J Fairbrother Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Kurt Deshayes Kurt Deshayes Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Donald S Kirkpatrick Donald S Kirkpatrick Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Domagoj Vucic Corresponding Author Domagoj Vucic Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Author Information Jasmin N Dynek1,‡, Tatiana Goncharov1,‡, Erin C Dueber1, Anna V Fedorova1, Anita Izrael-Tomasevic2, Lilian Phu2, Elizabeth Helgason1, Wayne J Fairbrother1, Kurt Deshayes1, Donald S Kirkpatrick2 and Domagoj Vucic 1 1Department of Protein Engineering, Genentech, Inc., South San Francisco, CA, USA 2Department of Protein Chemistry, Genentech, Inc., South San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. Department of Protein Engineering, Genentech, Inc. South San Francisco, 1 DNA Way, M/S 40, South San Francisco, CA 94080, USA. Tel.: +1 650 225 8839; Fax: +1 650 225 6127; E-mail: [email protected] The EMBO Journal (2010)29:4198-4209https://doi.org/10.1038/emboj.2010.300 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways. Introduction The regulated modification and degradation of cellular proteins by the ubiquitin-proteasome system are critical for modulation of many vital cellular processes in both normal and tumour cells (Hershko and Ciechanover, 1998). Ubiquitin, a protein whose C-terminus can be covalently linked to a lysine residue on a substrate, is instrumental in a number of cellular pathways including proinflammatory signalling, DNA damage response, and apoptosis (Hershko and Ciechanover, 1998). Ubiquitination occurs via a multistep reaction involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase (Schulman and Harper, 2009). RING domain-containing ubiquitin ligases bind to both the E2 and to the substrate proteins, and mediate transfer of the ubiquitin molecule from the E2 onto a lysine residue of the substrate protein (Deshaies and Joazeiro, 2009). In cells, a single ubiquitin molecule can be covalently attached to the substrate, termed monoubiquitination, or a substrate may be modified by polyubiquitin chains, involving additional ubiquitin–ubiquitin linkages (Pickart and Fushman, 2004). Seven lysine residues in each ubiquitin molecule present an opportunity for the assembly of diverse polyubiquitin chains, with varying cellular functions (Ikeda and Dikic, 2008; Komander, 2009). While K63-linked chains are mostly implicated in proinflammatory signalling, K48-linked polyubiquitin chains predominantly target proteins for proteasomal degradation (Hershko and Ciechanover, 1998; Glickman and Ciechanover, 2002; Komander, 2009). K11-linked chains have been less studied than K48 or K63 linkages, but they seem to serve as a degradation signal for APC/C substrates in the regulation of cell division (Kirkpatrick et al, 2006; Jin et al, 2008). Inhibitor of apoptosis (IAP) proteins are critical regulators of cellular survival capable of blocking apoptosis, modulating signal transduction, and affecting cellular proliferation (Salvesen and Duckett, 2002). In addition to their signature baculovirus IAP repeat (BIR) domains, several IAP proteins contain E3 ligase RING and ubiquitin-binding UBA domains (Vaux and Silke, 2005; Gyrd-Hansen et al, 2008; Blankenship et al, 2009). RING domain-containing cellular IAP1 and IAP2 (c-IAP1 and c-IAP2), XIAP and ML-IAP function as ubiquitin ligases that promote ubiquitination of themselves and several of their binding partners (Vaux and Silke, 2005). Cellular IAP proteins interact directly with tumour necrosis factor receptor-associated factor 2 (TRAF2), and via TRAF2, are recruited to TNF receptor-associated complexes, where they regulate apoptotic and NF-κB signalling (Rothe et al, 1995; Wang et al, 1998; Varfolomeev and Vucic, 2008). c-IAP1 and c-IAP2 are positive regulators of the TNFα-induced canonical NF-κB pathway, as they are required for receptor-interacting protein 1 (RIP1) ubiquitination and NF-κB activation (Bertrand et al, 2008; Mahoney et al, 2008; Varfolomeev et al, 2008). In the non-canonical NF-κB pathway, c-IAP proteins are negative regulators that ubiquitinate NF-κB inducing kinase (NIK), causing its proteasomal degradation and inhibition of signalling (Varfolomeev et al, 2007). RIP1 is an important link in TNFα-stimulated canonical NF-κB activation. Polyubiquitin chains assembled on RIP1 serve as a docking platform for the recruitment of the distal inhibitor of κB kinase (IKK) signalling complex, specifically the IKK subunit IKKγ, also known as NF-κB essential modifier (NEMO) (Kovalenko and Wallach, 2006; Li et al, 2006; Scheidereit, 2006). NEMO has been reported to have a high affinity for ubiquitin chains, particularly K63-linked polyubiquitin, as opposed to monoubiquitin (Ea et al, 2006; Wu et al, 2006). However, recent studies have shown that K63-linked ubiquitin chains might not be absolutely essential for TNFα-stimulated NF-κB activation, and that NEMO also possesses high affinity for other ubiquitin chains (Lo et al, 2009; Rahighi et al, 2009; Xu et al, 2009). There are over 30 human E2 ubiquitin-conjugating enzymes and possibly hundreds of E3 ubiquitin ligases. Thus, one can appreciate the degree of substrate specificity and ubiquitin chain variability that is potentially imparted by E2–E3 pairs. A previous study that interrogated E2–E3 interactions by a yeast two-hybrid strategy yielded insight into how specific ubiquitin modifications are generated by individual E2–E3 interactions (Christensen et al, 2007). Here, we describe a directed yeast two-hybrid screen that identified both novel and previously known E2 partners of IAP RING domains. Our results show that E2 enzymes of the UbcH5 family are critical functional partners for the E3 ligase activity of c-IAP proteins. Downregulation of UbcH5a/b/c levels restored c-IAP1 protein levels following destabilizing stimuli such as IAP antagonists or TWEAK or CD40 signalling, and prevented degradation of the c-IAP substrate NIK. In addition, we demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of RIP1. Using ubiquitin linkage-specific antibodies, we show that TNFα-stimulated NF-κB activation involves K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex, which is dependent on c-IAP1 and UbcH5. Finally, we provide evidence that the adaptor protein NEMO can bind K11 ubiquitin chains, indicating that this ubiquitin linkage may have a non-degradative signalling role in the activation of proliferative cellular pathways. Results Identification of interactions between IAP RING domains and E2 ubiquitin-conjugating enzymes IAP proteins c-IAP1, c-IAP2, ML-IAP, and XIAP are RING domain-containing ubiquitin ligases that promote assembly of polyubiquitin chains both on themselves and on several signalling molecules (Vaux and Silke, 2005; Varfolomeev and Vucic, 2008). To further elucidate the mechanism of ubiquitination mediated by IAP proteins, we examined interactions between IAP RING domains and E2 ubiquitin-conjugating enzymes in a series of directed yeast two-hybrid screens. We used individual c-IAP1, c-IAP2, ML-IAP, and XIAP RING domain constructs as baits against a library of 30 human E2 prey constructs. In total, 120 interactions were tested (Supplementary Figure S1). Positive and negative controls, including interactions of a BRCA1 RING–BARD1 RING fusion and UbcH5b/c, were assayed to validate the screen (Christensen et al, 2007) (Supplementary Figure S2). A number of E2 partners were shared amongst the IAP RING domains tested, while other E2 interactions were specific to particular IAP RING domains (Figure 1A; Supplementary Figure S1). Figure 1.IAP RING domain constructs interact with E2 ubiquitin-conjugating enzymes by yeast two-hybrid analysis. (A) Summary of positive interactions for each IAP RING domain DNA-binding fusion bait construct (c-IAP1 RING, c-IAP2 RING, ML-IAP RING, XIAP RING) with E2 activation domain fusion prey constructs. Interactions were scored according to growth on selective medium; + indicates growth present; − indicates growth absent. (B) Summary of interactions for UbcH5b and wild-type IAP RING domain prey constructs with wild-type IAP RING, IAP RING E2-binding surface mutants (c-IAP1 RING V573A, c-IAP2 RING V559A, ML-IAP RING V254A, XIAP RING I452A), and IAP RING dimerization mutants (c-IAP1 RING F616A, c-IAP2 RING F602A, ML-IAP RING F296A, XIAP RING 495A) bait constructs. WT IAP RING* denotes that the respective wild-type IAP RING prey constructs were tested for each set of interactions (for instance, wild-type c-IAP1 RING prey and c-IAP1 RING F616A bait). (C) Sequence alignment of IAP RING domains, bold black type indicates amino acid residues with conserved identities, bold grey type indicates amino acid residues with conserved side chain properties, and asterisks mark the locations of E2-binding surface and dimerization residues mutated in the IAP RING bait constructs. Download figure Download PowerPoint The three isoforms of the UbcH5 family of E2 enzymes (UbcH5a, UbcH5b, and UbcH5c) were found to interact with all IAP RING domains tested, consistent with published in vitro binding and ubiquitination assay studies (Figure 1A) (Yang and Du, 2004; Mace et al, 2008; Varfolomeev et al, 2008). The RING domain of c-IAP1 specifically interacted with tsg101, Ube2s, and Rad6b, while Ube2Q2 interacted with the RING domains of c-IAP1 and c-IAP2, but not with those of ML-IAP or XIAP (Figure 1A). On the other hand, ML-IAP and XIAP RING domains interacted with UbcH6, again consistent with published in vitro ubiquitination assay results (Yang and Du, 2004). Although UbcH13 has been reported to function as an E2 in combination with c-IAP in in vitro ubiquitination assays (Bertrand et al, 2008), interactions were not observed between UbcH13 and any of the IAP RING family members tested (Supplementary Figure S1). This finding is consistent with our previously published in vitro ubiquitination assay data (Varfolomeev et al, 2008). To further verify this result, the UbcH13 construct was functionally validated in yeast two-hybrid assays with TRAF2 and TRAF6 RING domains. As previously reported (Yin et al, 2009a, 2009b), TRAF6 RING domain bound UbcH13, while no interaction was observed between TRAF2 RING and UbcH13 (Supplementary Figure S2). In order to further validate the IAP RING domain interactions with the UbcH5 family, we made mutations in the IAP RING bait constructs that are predicted to disrupt the RING domain's E2-binding surface or to prevent dimerization (Figure 1B and C) (Mace et al, 2008). In agreement with the reported structural studies, we found that the c-IAP2 V559A E2-binding surface mutant lost the ability to interact with UbcH5b. However, it retained the ability to dimerize, as assayed by interaction with a wild-type c-IAP2 RING prey construct (Figure 1B). On the other hand, the c-IAP2 F602A dimerization mutant failed to interact with UbcH5b and was unable to dimerize with the wild-type c-IAP2 RING domain. The c-IAP1, ML-IAP, and XIAP E2-binding surface mutations also abrogated interactions with UbcH5b, and, except in the case of XIAP RING I452A, had no effect on RING domain dimerization. Predicted dimerization mutations in c-IAP1, ML-IAP, and XIAP RING domains prevented their interaction with the corresponding wild-type IAP RING domain constructs. The ML-IAP RING F296A dimerization mutant did not interact with UbcH5b, but the c-IAP1 and XIAP dimerization mutants supported interactions with UbcH5b (Figure 1B). Additionally, mutations of the predicted E2 binding and RING domain dimerization residues in c-IAP1 and ML-IAP RING domains prevented their interactions with several other E2 enzymes identified as potential IAP-interacting partners from the initial yeast two-hybrid screen (Supplementary Figure S3A and B). We also tested the Ubc9 interactions in an analogous manner and concluded that the observed Ubc9 interactions (Figure 1A) were most likely non-specific, as none of the mutations tested affected interaction with Ubc9 (Supplementary Figure S3C). In sum, our directed yeast two-hybrid screens confirmed several known interactions and also identified a number of novel interactions, between the IAP RING domains and E2 enzymes, thereby providing a more thorough understanding of IAP-mediated ubiquitination. Ube2S promotes ubiquitin chain extension in combination with c-IAP1 and UbcH5a Having identified Ube2S as a binding partner of the c-IAP1 RING domain in a directed yeast two-hybrid screen, we wanted to investigate whether this E2 enzyme can work with the E3 ligase c-IAP1 to promote ubiquitin chain formation. Initial attempts using a standard ubiquitination protocol with Ube2S and c-IAP1, together with an E1 enzyme and an energy source, did not yield any ubiquitin chains at several different temperatures (17–37°C) and reaction times (30 min to 2 h) (Figure 2A and B). At the same time, UbcH5a in combination with c-IAP1 efficiently formed polyubiquitin chains. This validates the other components of the reaction, including the recombinant c-IAP1 protein (Figure 2A and B). Recent reports on the enzymatic activity of Ube2S indicate that this E2 enzyme can extend the ubiquitin chains initiated by other E2 enzymes, such as UbcH10 (Garnett et al, 2009; Williamson et al, 2009). Thus, we modified the reactions to include a 5-min preincubation of c-IAP1 with 5% of the UbcH5a concentration used in UbcH5a control reactions as a first step, which did not promote significant c-IAP1 autoubiquitination. Following that first step, the E2 enzymes UbcH5a or Ube2S were added, and these second-step reactions were allowed to proceed for another 35 min. This experimental design revealed ubiquitination activity and demonstrated the ability of Ube2S to promote ubiquitin chain assembly in conjunction with UbcH5a (Figure 2). Ubiquitination reactions were quantified using the ubiquitin-AQUA method (Kirkpatrick et al, 2006; Blankenship et al, 2009). Equivalent gel regions from each sample were excised from the Coomassie blue-stained SDS–PAGE gel, beginning above the unmodified c-IAP1 bands (Figure 2A). Quantification of the ubiquitin reactions confirmed the results obtained using an anti-ubiquitin antibody in western blotting, and demonstrated the ability of Ube2S to promote ubiquitin chain assembly (Figure 2C; Supplementary Figure S4). Thus, Ube2S can act as a functional partner of c-IAP1 to promote the assembly of polyubiquitin chains in combination with another E2 enzyme, namely UbcH5a. Figure 2.Ube2S can promote ubiquitin chain extension in combination with c-IAP1 and UbcH5a. (A) Coomassie-stained gel containing equivalent amounts of in vitro c-IAP1 autoubiquitination reactions (rxn) performed in two steps with the first-step reaction conducted in the absence or the presence of UbcH5a as described in Materials and methods. The red boxes mark the boundaries of gel regions that were excised and subjected to in-gel trypsin digestion, followed by Ubiquitin-AQUA analysis. (B) Western blot using anti-ubiquitin antibody (P4D1) of autoubiquitination reactions. (C) Quantification of the amount of total ubiquitin in each corresponding gel region for each sample. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint The UbcH5 family of E2 enzymes regulates the stability of c-IAP1 and the c-IAP substrate NIK We next focussed on the c-IAP1 E2 interactions, both those shared amongst IAP family members and those specific to c-IAP1, to investigate their physiological impact on the stability of c-IAP1 protein and its substrates in human cancer cells. To this end, we transiently transfected HKB11 cells stably expressing the human CD40 receptor (HKB11-CD40) with siRNAs targeting the c-IAP1-interacting E2 enzymes. Treatment of HKB11 cells with anti-CD40 antibody resulted in a decrease in c-IAP1 protein levels, as seen in control siRNA-transfected cells (Figure 3A). Knockdown of the UbcH5 family of E2 enzymes stabilized c-IAP1 protein levels following CD40 stimulation (Figure 3A, top left panel), while no changes in c-IAP1 protein levels were observed with siRNA knockdowns of Ube2S, tsg101, Ube2Q2, or Rad 6B (Figure 3A; Supplementary Table I). The stability of XIAP was not affected by CD40 treatment while c-IAP2 or ML-IAP expression could not be detected in these cells (Supplementary Figure S5A). The IAP antagonist MV1 stimulates the E3 ligase activity and autoubiquitination of c-IAP1 (Varfolomeev et al, 2007). However, c-IAP1 protein levels were stabilized in cells transfected with UbcH5 siRNA following MV1 treatment (Supplementary Figure S5B). We also analysed the role of UbcH5 proteins in signalling by the TNF superfamily ligand, TWEAK. Downregulation of UbcH5 family expression, but not Ube2S expression, stabilized c-IAP1 protein levels following TWEAK treatment (Figure 3B). In addition, UbcH5 knockdown significantly blunted stimulation of gene expression by TWEAK and TNFα (Figure 3C; Supplementary Figure S6), suggesting that the UbcH5 family of E2 enzymes has an important role in these TNF ligands-mediated signalling pathways. Figure 3.siRNA knockdown of the UbcH5 family increases the stability of c-IAP1 and the c-IAP1 substrate NIK. (A) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases c-IAP1 protein levels following CD40 treatment. HKB11-CD40 cells were transfected with control siRNA, or siRNA targeting the UbcH5 family (UbcH5a, UbcH5b, UbcH5c isoforms), Ube2S, tsg101, Ube2Q2, or Rad6B for 48 h. Anti-CD40 antibody was cross-linked with secondary antibody for 10 min, and then added to cells at a concentration of 0.5 μg/ml for 0, 5, or 30 min. Lysates were collected and subjected to western blot analysis using antibodies for detection of c-IAP1, UbcH5 (the antibody recognizes all three isoforms), tsg101, and actin, as a loading control. In parallel, RNA samples were collected and purified to monitor the efficiency of Ube2Q2 and Rad6B siRNA-knockdown by quantative RT–PCR analysis (Supplementary Table I). (B) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases c-IAP1 protein levels following TWEAK treatment. HT1080 cells were transfected with control siRNA, or siRNA targeting the UbcH5 family (UbcH5a, UbcH5b, UbcH5c isoforms), or Ube2S for 48 h and then treated with TWEAK (100 ng/ml) for indicated time points. Lysates were collected and subjected to western blot analysis using antibodies for detection of c-IAP1, UbcH5 (the antibody recognizes all three isoforms), Ube2S, and actin, as a loading control. (C) Knockdown of UbcH5 family inhibits gene induction by TWEAK. HT1080 cells were treated with TWEAK for 4 h (or 7 h for RelB), and RNA samples from treated and untreated cells were analysed by quantitative real-time PCR analysis. All values were normalized to an RPL19 RNA internal control. Columns represent mean from triplicate experiments and bars represent s.d. (D) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases NIK protein stability in the presence of the c-IAP proteins. HEK293T cells were transfected with control siRNA, or siRNA targeting the UbcH5 family, Ube2S, tsg101, Ube2Q2, or Rad6B. At 24 h later, cells were transfected with myc-NIK1 plus FLAG-c-IAP1, FLAG-c-IAP2, or FLAG-vector. The following day, lysates were collected and analysed by western blot, and RNA was prepared for quantitative RT–PCR analysis (Supplementary Table II) as in panel (A). Download figure Download PowerPoint NIK is a substrate of the E3 ligases c-IAP1 and c-IAP2 in the non-canonical NF-κB signalling pathway (Varfolomeev et al, 2007; Vince et al, 2007). In order to examine which E2 enzyme acts in concert with the c-IAP proteins to regulate NIK stability we co-expressed myc-NIK and FLAG-c-IAP1, FLAG-c-IAP2, or FLAG-vector in 293T cells, in combination with transfections of siRNA targeting the c-IAP1-interacting E2 enzymes. NIK protein levels decreased drastically upon co-expression with either c-IAP1 or c-IAP2, as shown previously (Varfolomeev et al, 2007). However, siRNA knockdown of the UbcH5 family greatly increased NIK protein levels in cells overexpressing c-IAP1 or c-IAP2 (Figure 3D, left panel). NIK protein levels were largely unchanged when any of the other c-IAP1-interacting E2 enzymes were silenced via siRNA (Figure 3D; Supplementary Table II). In addition, E2 binding and RING dimerization mutants of c-IAP1 were unable to promote NIK degradation, while the co-expression of siRNA-resistant UbcH5b construct reversed the effect of UbcH5 knockdown on NIK stability (Supplementary Figure S7A and B). Collectively, these results suggest that the UbcH5 family of E2 enzymes regulates the stability of both c-IAP1 protein and its substrates in response to cellular signalling. c-IAP1 promotes K11 polyubiquitin linkage formation on RIP1 Another important substrate of the c-IAP proteins is the Ser/Thr kinase RIP1, a critical mediator of TNFα-stimulated canonical NF-κB signalling (Varfolomeev and Vucic, 2008). Cellular IAP proteins can promote K48- and K63-linked polyubiquitination of RIP1 (Bertrand et al, 2008; Varfolomeev et al, 2008), but whether RIP1 can be modified by other ubiquitin linkages is not clear. Given that c-IAP1 autoubiquitination generates significant levels of K11 linkages in vitro (Blankenship et al, 2009) and that the c-IAP1 RING domain interacts with the K11 linkage-promoting E2 enzymes UbcH5 and Ube2s, we examined whether c-IAP1 can promote addition of K11 linkages to RIP1. First, we demonstrated that c-IAP1 is capable of mediating K11-linked polyubiquitination on itself and RIP1. Transfection of 293T cells with c-IAP1, RIP1 and wild-type or K11-, K48-, or K63-only ubiquitin mutants, followed by lysis under denaturing conditions and immunoprecipitation showed that c-IAP1 and RIP1 can carry K11 linkages in vivo (Figure 4A). On the other hand, the E2 binding and RING dimerization mutants of c-IAP1 failed to promote RIP1 ubiquitination in 293T cells or c-IAP1-deficient MEF cells (Supplementary Figure S8A and B). Reconstituted ubiquitination reactions with recombinant proteins demonstrated the K11-linked polyubiquitination of c-IAP1 and RIP1 in vitro in combination with UbcH5 a, b, or c variants and with K11R, K48R, or K63R ubiquitin proteins as well as with Ube2S in a two-step reaction (Figure 4B; Supplementary Figure S8C–E). The closely related c-IAP2 protein was also capable of mediating K11-linked polyubiquitination of itself and of RIP1 (Supplementary Figure S9). Figure 4.c-IAP1 promotes K11 polyubiquitin linkage formation on RIP1. (A) Ectopic expression of c-IAP1 and RIP1 promotes K11-linked polyubiquitination in 293T cells. HEK293T cells were transfected with myc-RIP1, HA-tagged ubiquitin constructs (WT, K11-, K48-, or K63-only), with or without Flag-c-IAP1 for 24 h. Cells were pretreated with MG132 (20 μM) for 1 h, lysates were boiled in NP40 lysis buffer containing 1% SDS for 10 min, diluted 10-fold and immunoprecipitated using anti-myc or anti-Flag beads. Flag c-IAP1 and myc-RIP1 were detected in immunoprecipitated material and in input lysates using anti-HA, anti-myc, and anti-Flag antibodies. (B) Recombinant RIP1 was incubated for 45 min in a ubiquitination reaction in the absence or the presence of recombinant c-IAP1, UbcH5a, and wild-type or K11-only ubiquitin proteins. RIP1 and c-IAP1 modifications were determined with anti-RIP1 and anti-c-IAP1 antibodies. (C) Determination of c-IAP1-mediated polyubiquitin linkages on RIP1. Recombinant RIP1 (5 μg) was incubated for 45 min in ubiquitination reactions in the presence of recombinant c-IAP1 (1.25 μg), UbcH5a (1.25 μg), and wild-type ubiquitin. Following ubiquitination reactions, samples were incubated in 6 M Urea at room temperature for 30 min with rocking, diluted 15 times and immunoprecipitated using anti-RIP1 antibodies. Coomassie-stained gel containing equivalent amounts of immunoprecipitated RIP1 (left side): the red lines mark the boundaries of gel regions that were excised and subjected to in-gel trypsin digestion, followed by Ubiquitin-AQUA analysis using OrbiTrap (bottom and right). Extracted ion chromatograms of indicated peptides from gel region A are shown where heavy peaks (red), corresponding to isotope labelled internal standard peptides, and light peaks (blue), corresponding to digested analyte peptides, are labelled with retention time and m/z ratio. The relative abundance for light peptide chromatograms (y axis) has been zoomed 2X relative to corresponding heavy peptides. Quantification of the amount of c-IAP1, RIP1 protein, total ubiquitin, and K11-linked ubiquitin for each corresponding gel region for each sample is shown on the bottom. Download figure Download PowerPoint Next, we further explored whether c-IAP1 could promote K11-linked RIP1 ubiquitination using quantitative mass spectrometry. To

<h2>Summary</h2> The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. <i>hul5</i> mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A <i>hul5</i> null mutation suppresses a <i>ubp6</i> null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.

The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.

<h2>Summary</h2> Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.

Mass-spectrometry-based proteomics has become an essential tool for the qualitative and quantitative analysis of cellular systems. The biochemical complexity and functional diversity of the ubiquitin system are well suited to proteomic studies. This review summarizes advances involving the identification of ubiquitinated proteins, the elucidation of ubiquitin-modification sites and the determination of polyubiquitin chain linkages, as well as offering a perspective on the application of emerging technologies for mechanistic and functional studies of protein ubiquitination.

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger 4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.

Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin–proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults.

Cell cycle progression requires the E3 ubiquitin ligase anaphase-promoting complex (APC/C), which uses the substrate adaptors CDC20 and CDH1 to target proteins for proteasomal degradation. The APCCDH1 substrate cyclin A is critical for the G1/S transition and, paradoxically, accumulates even when APCCDH1 is active. We show that the deubiquitinase USP37 binds CDH1 and removes degradative polyubiquitin from cyclin A. USP37 was induced by E2F transcription factors in G1, peaked at G1/S, and was degraded in late mitosis. Phosphorylation of USP37 by CDK2 stimulated its full activity. USP37 overexpression caused premature cyclin A accumulation in G1 and accelerated S phase entry, whereas USP37 knockdown delayed these events. USP37 was inactive in mitosis because it was no longer phosphorylated by CDK2. Indeed, it switched from an antagonist to a substrate of APCCDH1 and was modified with degradative K11-linked polyubiquitin.

The anaphase-promoting complex or cyclosome (APC/C) initiates mitotic exit by ubiquitylating cell-cycle regulators such as cyclin B1 and securin. Lys 48-linked ubiquitin chains represent the canonical signal targeting proteins for degradation by the proteasome, but they are not required for the degradation of cyclin B1. Lys 11-linked ubiquitin chains have been implicated in degradation of APC/C substrates, but the Lys 11-chain-forming E2 UBE2S is not essential for mitotic exit, raising questions about the nature of the ubiquitin signal that targets APC/C substrates for degradation. Here we demonstrate that multiple monoubiquitylation of cyclin B1, catalysed by UBCH10 or UBC4/5, is sufficient to target cyclin B1 for destruction by the proteasome. When the number of ubiquitylatable lysines in cyclin B1 is restricted, Lys 11-linked ubiquitin polymers elaborated by UBE2S become increasingly important. We therefore explain how a substrate that contains multiple ubiquitin acceptor sites confers flexibility in the requirement for particular E2 enzymes in modulating the rate of ubiquitin-dependent proteolysis.

The proteasome recognizes its substrates via a diverse set of ubiquitin receptors, including subunits Rpn10/S5a and Rpn13. In addition, shuttling factors, such as Rad23, recruit substrates to the proteasome by delivering ubiquitinated proteins. Despite the increasing understanding of the factors involved in this process, the regulation of substrate delivery remains largely unexplored. Here we report that Rpn10 is monoubiquitinated in vivo and that this modification has profound effects on proteasome function. Monoubiquitination regulates the capacity of Rpn10 to interact with substrates by inhibiting Rpn10's ubiquitin-interacting motif (UIM). We show that Rsp5, a member of NEDD4 ubiquitin-protein ligase family, and Ubp2, a deubiquitinating enzyme, control the levels of Rpn10 monoubiquitination in vivo. Notably, monoubiquitination of Rpn10 is decreased under stress conditions, suggesting a mechanism of control of receptor availability mediated by the Rsp5-Ubp2 system. Our results reveal an unanticipated link between monoubiquitination signal and regulation of proteasome function.

Epithelial to mesenchymal transition (EMT) is a key process in embryonic development and has been associated with cancer metastasis and drug resistance. For example, in EGFR mutated non-small cell lung cancers (NSCLC), EMT has been associated with acquired resistance to the EGFR inhibitor erlotinib. Moreover, "EGFR-addicted" cancer cell lines induced to undergo EMT become erlotinib-resistant in vitro. To identify potential therapeutic vulnerabilities specifically within these mesenchymal, erlotinib-resistant cells, we performed a small molecule screen of ~200 established anti-cancer agents using the EGFR mutant NSCLC HCC827 cell line and a corresponding mesenchymal derivative line. The mesenchymal cells were more resistant to most tested agents; however, a small number of agents showed selective growth inhibitory activity against the mesenchymal cells, with the most potent being the Abl/Src inhibitor, dasatinib. Analysis of the tyrosine phospho-proteome revealed several Src/FAK pathway kinases that were differentially phosphorylated in the mesenchymal cells, and RNAi depletion of the core Src/FAK pathway components in these mesenchymal cells caused apoptosis. These findings reveal a novel role for Src/FAK pathway kinases in drug resistance and identify dasatinib as a potential therapeutic for treatment of erlotinib resistance associated with EMT.

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery—the ubiquitin-proteasome system and the ubiquitin trafficking system—were unevenly perturbed by expression of K0 ubiquitin. Post-translational modification of cellular proteins with ubiquitin determines their fate by influencing protein-protein interactions, altering recognition, targeting to cellular compartments, or by promoting their degradation at the 26S proteasomes (1Hicke L. Dunn R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins.Annu. Rev. Cell Dev. 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The majority of proteasome substrates are tagged not by a single ubiquitin (monoUb) 1The abbreviations used are:monoUbsingle ubiquitinpolyUbpolyubiquitinUbubiquitinWCEwhole cell extractSILACstable isotope labeling with amino acids in cell cultureMWmolecular weightERADER-associated degradation., but by a polyubiquitin (polyUb) chain. Lys48 is the only lysine on ubiquitin whose substitution to arginine is lethal, pointing to a unique and essential role for Lys48-linked chains (21Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.Mol. Cellular Biol. 1994; 14: 5501-5509Crossref PubMed Google Scholar). It is generally thought that such Lys48-linked polyUb chains longer than four ubiquitin molecules are the preferred signal for efficient recognition and degradation by 26S proteasomes (22Thrower J.S. Hoffman L. Rechsteiner M. Pickart C.M. Recognition of the polyubiquitin proteolytic signal.EMBO J. 2000; 19: 94-102Crossref PubMed Google Scholar). Once bound by proteasomes, substrate-conjugates are deubiquitinated, unfolded, and subsequently degraded. single ubiquitin polyubiquitin ubiquitin whole cell extract stable isotope labeling with amino acids in cell culture molecular weight ER-associated degradation. Other biological pathways that are regulated by ubiquitination include endocytosis and intracellular trafficking (23Madshus I.H. Ubiquitin binding in endocytosis–how tight should it be and where does it happen?.Traffic. 2006; 7: 258-261Crossref PubMed Scopus (0) Google Scholar, 24Mosesson Y. Yarden Y. Monoubiquitylation: a recurrent theme in membrane protein transport.Isr. Med. Assoc. J. 2006; 8: 233-237PubMed Google Scholar, 25Saksena S. Sun J. Chu T. Emr S.D. ESCRTing proteins in the endocytic pathway.Trends Biochem. 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Lys63-linked polyubiquitin chains: linking more than just ubiquitin.Cancer Biol. Ther. 2006; 5: 1273-1274Crossref PubMed Scopus (0) Google Scholar, 31Haglund K. Dikic I. Ubiquitylation and cell signaling.EMBO J. 2005; 24: 3353-3359Crossref PubMed Scopus (541) Google Scholar). Most of these nonproteolytic roles are carried out by so called “alternative” ubiquitin signals, such as embodied by conjugation of a single ubiquitin molecule (monoubiquitination), or by non-Lys48-linked polyUb chains. For instance, nonproteolytic processes associated with Lys63 chains have been documented in protein trafficking, DNA damage tolerance, the inflammatory response, and ribosomal protein function (4Pickart C.M. Fushman D. Polyubiquitin chains: polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (777) Google Scholar). In protein trafficking, Lys63-linked polyUb serves as a signal mediating the internalization of plasma membrane receptors and transporters, intracellular transport, and subsequent lysosomal and vacuolar degradation (32Duncan L.M. Piper S. Dodd R.B. Saville M.K. Sanderson C.M. Luzio J.P. Lehner P.J. Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules.EMBO J. 2006; 25: 1635-1645Crossref PubMed Scopus (200) Google Scholar, 33Paiva S. Vieira N. Nondier I. Haguenauer-Tsapis R. Casal M. Urban-Grimal D. Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of lysine 63-linked ubiquitin chains.J. Biol. Chem. 2009; 284: 19228-19236Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 34Barriere H. Nemes C. Du K. Lukacs G.L. Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery.Mol. Biol. 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Agonist-promoted Lys63-linked polyubiquitination of the human kappa-opioid receptor is involved in receptor down-regulation.Mol. Pharmacol. 2008; 73: 1319-1330Crossref PubMed Scopus (0) Google Scholar, 39Varghese B. Barriere H. Carbone C.J. Banerjee A. Swaminathan G. Plotnikov A. Xu P. Peng J. Goffin V. Lukacs G.L. Fuchs S.Y. Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway.Mol. Cell. Biol. 2008; 28: 5275-5287Crossref PubMed Scopus (62) Google Scholar). Lys63-linked chains function also as a trafficking signal at the endosomal level for MVB sorting (40Lauwers E. Erpapazoglou Z. Haguenauer-Tsapis R. André B. The ubiquitin code of yeast permease trafficking.Trends Cell Biol. 2010; 20: 196-204Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). By promoting Lys63-linked polymerization, Rsp5, an E3 ubiquitin ligase of the Nedd4 HECT family, has been shown to have a prevailing role in ubiquitination of plasma-membrane transporters in yeast cells (41Belgareh-Touzé N. Léon S. Erpapazoglou Z. Stawiecka-Mirota M. Urban-Grimal D. Haguenauer-Tsapis R. Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking.Biochem. Soc. Trans. 2008; 36: 791-796Crossref PubMed Scopus (0) Google Scholar, 42Kee Y. Muñoz W. Lyon N. Huibregtse J.M. The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys63-linked polyubiquitin conjugates in Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 36724-36731Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 43Kim H.C. Huibregtse J.M. Polyubiquitination by HECT E3s and the determinants of chain type specificity.Mol. Cell. Biol. 2009; 29: 3307-3318Crossref PubMed Scopus (164) Google Scholar, 44Saeki Y. Kudo T. Sone T. Kikuchi Y. Yokosawa H. Toh-e A. Tanaka K. Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome.EMBO J. 2009; 28: 359-371Crossref PubMed Scopus (176) Google Scholar). In most cases, this ligase does not bind its substrates directly, but is recruited to them via a subset of specific adaptors some of which were shown to undergo Rsp5-dependent ubiquitination themselves (40Lauwers E. Erpapazoglou Z. Haguenauer-Tsapis R. André B. The ubiquitin code of yeast permease trafficking.Trends Cell Biol. 2010; 20: 196-204Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 45Léon S. Haguenauer-Tsapis R. Ubiquitin ligase adaptors: regulators of ubiquitylation and endocytosis of plasma membrane proteins.Exp. Cell Res. 2009; 315: 1574-1583Crossref PubMed Scopus (75) Google Scholar, 46Lin C.H. MacGurn J.A. Chu T. Stefan C.J. Emr S.D. Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface.Cell. 2008; 135: 714-725Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 47Shearwin-Whyatt L. Dalton H.E. Foot N. Kumar S. Regulation of functional diversity within the Nedd4 family by accessory and adaptor proteins.Bioessays. 2006; 28: 617-628Crossref PubMed Scopus (122) Google Scholar). Complicating the ability to attribute specific functions to Lys63 linkages, many membrane processes associated with Lys63 linkages are also driven by monoubiquitination. Often the same substrates are documented to be targets of both Lys63 polyUb or monoUb modifications. Both signals may partially overlap, though mild discrepancies between samples may also arise from rapid deubiquitination of Lys63 chains (48Cooper E.M. Cutcliffe C. Kristiansen T.Z. Pandey A. Pickart C.M. Cohen R.E. K63-specific deubiquitination by two JAMM/MPN+ complexes: BRISC-associated Brcc36 and proteasomal Poh1.EMBO J. 2009; 28: 621-631Crossref PubMed Scopus (161) Google Scholar, 49Sims J.J. Cohen R.E. Linkage-specific avidity defines the lysine 63-linked polyubiquitin-binding preference of rap80.Mol. Cell. 2009; 33: 775-783Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Notably Ubp2, specifically deubiquitinates Lys63-linked ubiquitin chains on Rsp5-substrates leading to an increase in monoubiquitinated substrates (42Kee Y. Muñoz W. Lyon N. Huibregtse J.M. The deubiquitinating enzyme Ubp2 modulates Rsp5-dependent Lys63-linked polyubiquitin conjugates in Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 36724-36731Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Sample preparation may thus influence efficiency of trapping of polyUb chains, in particular the relatively labile Lys63 linkages. Understanding the relative prevalence and relative efficiency of monoubiquitination versus Lys63 chains in intracellular transport and endocytosis is a subject of intense investigation and great scrutiny. The precise biological differences between polyUb chains of various topologies have not been broadly understood yet, though sporadic observations keep coming in. Recent studies have spotlighted Lys11-linked ubiquitin chains, revealing their involvement in endoplasmic reticulum associated degradation (ERAD) as modifiers of the E2 Ubc6, which has also been proposed to participate in the synthesis of these chains (8Xu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (753) Google Scholar). Other studies have related Lys11 chains to regulation of mitotic protein degradation and cell cycle control (50Kirkpatrick D.S. Hathaway N.A. Hanna J. Elsasser S. Rush J. Finley D. King R.W. Gygi S.P. Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.Nat. Cell Biol. 2006; 8: 700-710Crossref PubMed Scopus (325) Google Scholar, 51Matsumoto M.L. Wickliffe K.E. Dong K.C. Yu C. Bosanac I. Bustos D. Phu L. Kirkpatrick D.S. Hymowitz S.G. Rape M. Kelley R.F. Dixit V.M. K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody.Mol. Cell. 2010; Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Lys6 chains were shown to be synthesized by the BRCA1/BARD1 complex (52Nishikawa H. Ooka S. Sato K. Arima K. Okamoto J. Klevit R.E. Fukuda M. Ohta T. Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase.J. Biol. Chem. 2004; 279: 3916-3924Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 53Wu-Baer F. Lagrazon K. Yuan W. Baer R. The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin.J. Biol. Chem. 2003; 278: 34743-34746Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar); Lys27 chains were described to be involved in ubiquitination of the transcription factor Jun, a modification required for its unconventional lysosomal targeting (54Ikeda H. Kerppola T.K. Lysosomal localization of ubiquitinated Jun requires multiple determinants in a lysine-27-linked polyubiquitin conjugate.Mol. Biol. Cell. 2008; 19: 4588-4601Crossref PubMed Google Scholar); Lys33 chains were recently reported to modify TCR-ζ thereby regulating cell-surface-receptor-mediated signal transduction; involvement of Lys29 chains was demonstrated in protein degradation and recruitment of a chain elongating factor E4 (55Johnson E.S. Ma P.C. Ota I.M. Varshavsky A. A proteolytic pathway that recognizes ubiquitin as a degradation signal.J. Biol. Chem. 1995; 270: 17442-17456Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 56Mastrandrea L.D. You J. Niles E.G. Pickart C.M. E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.J. Biol. Chem. 1999; 274: 27299-27306Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Two different substrates of AIP4/Itch were also described to be modified by K29-linked ubiquitin chains in the context of Notch trafficking and signaling (57Chastagner P. Israël A. Brou C. AIP4/Itch regulates Notch receptor degradation in the absence of ligand.PLoS One. 2008; 3e2735 Crossref PubMed Scopus (97) Google Scholar). Adding yet another layer of complexity to the conjugated ubiquitin landscape, there are additional modifications by ubiquitin, including: mono- and multiple-mono ubiquitinations (58Haglund K. Sigismund S. Polo S. Szymkiewicz I. Di Fiore P.P. Dikic I. Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation.Nat. Cell Biol. 2003; 5: 461-466Crossref PubMed Scopus (627) Google Scholar, 59Hicke L. Protein regulation by monoubiquitin.Nat. Rev. Mol. Cell Biol. 2001; 2: 195-201Crossref PubMed Scopus (941) Google Scholar, 60Hicke L. A New Ticket for Entry into Budding Vesicles–Ubiquitin.Cell. 2001; 106: 527-530Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar), mixed or branched ubiquitin chains (61Ziv I. Kleifeld O. Glickman M. Nonconformity in ubiquitin compliance.EMBO J. 2009; 28: 1825-1827Crossref PubMed Scopus (2) Google Scholar, 62Kim H.T. Kim K.P. Lledias F. Kisselev A.F. Scaglione K.M. Skowyra D. Gygi S.P. Goldberg A.L. Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages.J. Biol. Chem. 2007; 282: 17375-17386Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 63Uchiki T. Kim H.T. Zhai B. Gygi S.P. Johnston J.A. O'Bryan J.P. Goldberg A.L. The ubiquitin-interacting motif protein, S5a, is ubiquitinated by all types of ubiquitin ligases by a mechanism different from typical substrate recognition.J. Biol. Chem. 2009; 284: 12622-12632Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 64Ben-Saadon R. Zaaroor D. Ziv T. Ciechanover A. 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Alternatively, polymerization quality control may be less rigidly enforced generating mixed signals in these pathways. Utilization of ubiquitin mutants is a strategy used to study the role or different ubiquitin chains. Substituting lysine residues in the ubiquitin molecule with arginine blocks specific chain extensions and thereby inhibits downstream consequences associated with a specific ubiquitin linkage (67Volk S. Wang M. Pickart C.M. Chemical and genetic strategies for manipulating polyubiquitin chain structure.Methods Enzymol. 2005; 399: 3-20Crossref PubMed Scopus (18) Google Scholar). Single-lysine ubiquitin mutants and a lysine-less, nonextendable mutant (K0 Ub) (68Arnason T. Ellison M.J. Stress resistance in Saccharomyces cerevisiae is strongly correlated with assembly of a novel type of multiubiquitin chain.Mol. Cell. 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Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia–reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia–reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.

•Ubiquitin ligase COP1 promotes proteasomal degradation of c/EBPβ•Loss of COP1 triggers a pro-inflammatory gene expression program in microglia•COP1-deficient microglia exhibit c/EBPβ- and C1q-dependent neurotoxicity•COP1-deficient microglia exacerbate Tau-driven pathology in mice SummaryDysregulated microglia are intimately involved in neurodegeneration, including Alzheimer’s disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPβ in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPβ accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPβ-dependent gene expression programs in microglia. Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer’s disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPβ) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPβ in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPβ accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPβ-dependent gene expression programs in microglia.

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.

Abstract PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. Significance: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors. See related commentary by Vanhaesebroeck et al., p. 20. This article is highlighted in the In This Issue feature, p. 1

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain.

Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine 48-linked ubiquitin is essential for marking targets for proteasomal degradation, but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine 48 and lysine 63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine 48 linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.

Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid–induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss.

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSimplified wet ash procedure for total phosphorus analysis of organophosphonates in biological samplesDonald S. Kirkpatrick and Stephen H. BishopCite this: Anal. Chem. 1971, 43, 12, 1707–1709Publication Date (Print):October 1, 1971Publication History Published online1 May 2002Published inissue 1 October 1971https://doi.org/10.1021/ac60306a046RIGHTS & PERMISSIONSArticle Views279Altmetric-Citations67LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (381 KB) Get e-Alerts Get e-Alerts

The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3β to foster phosphorylation of the Wnt effector β-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced β-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling. <b>Background:</b> APC is modified with Lys-63-linked polyubiquitin when bound to Axin in an assembled β-catenin destruction complex. <b>Results:</b> HectD1 E3 ligase modifies APC with Lys-63-linked ubiquitin chains to facilitate the APC-Axin interaction. <b>Conclusion:</b> HectD1 is a candidate E3 ligase for APC. <b>Significance:</b> The identification of HectD1 could lead to a better understanding of APC function.

We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.

GDC-0834, a Bruton’s tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (<i>R</i>)-<i>N</i>-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[<i>b</i>] thiophene-2-carboxamide) was rapidly hydrolyzed with a CL_int of 0.511 ml/min per milligram of protein. Aldehyde oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC₅₀ values ranging from 0.86 to 1.87 <i>μ</i>M. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834.

Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.

High-throughput genomic and proteomic studies have generated near-comprehensive catalogs of biological constituents within many model systems. Nevertheless, static catalogs are often insufficient to fully describe the dynamic processes that drive biology. Quantitative proteomic techniques address this need by providing insight into closely related biological states such as the stages of a therapeutic response or cellular differentiation. The maturation of quantitative proteomics in recent years has brought about a variety of technologies, each with their own strengths and weaknesses. It can be difficult for those unfamiliar with this evolving landscape to match the experiment at hand with the best tool for the job. Here, we outline quantitative methods for proteomic mass spectrometry and discuss their benefits and weaknesses from the perspective of the biologist aiming to generate meaningful data and address mechanistic questions. High-throughput genomic and proteomic studies have generated near-comprehensive catalogs of biological constituents within many model systems. Nevertheless, static catalogs are often insufficient to fully describe the dynamic processes that drive biology. Quantitative proteomic techniques address this need by providing insight into closely related biological states such as the stages of a therapeutic response or cellular differentiation. The maturation of quantitative proteomics in recent years has brought about a variety of technologies, each with their own strengths and weaknesses. It can be difficult for those unfamiliar with this evolving landscape to match the experiment at hand with the best tool for the job. Here, we outline quantitative methods for proteomic mass spectrometry and discuss their benefits and weaknesses from the perspective of the biologist aiming to generate meaningful data and address mechanistic questions. Throughout the pioneering days of genomic and proteomic research, much effort was put into constructing comprehensive catalogs of biological data, exemplified by the sequencing of the human genome (1Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (16119) Google Scholar, 2Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. et al.The sequence of the human genome.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (9791) Google Scholar) and proteome (3Kim M.-S. Pinto S.M. Getnet D. Nirujogi R.S. Manda S.S. Chaerkady R. Madugundu A.K. Kelkar D.S. Isserlin R. Jain S. Thomas J.K. Muthusamy B. Leal-Rojas P. Kumar P. Sahasrabuddhe N.A. et al.A draft map of the human proteome.Nature. 2014; 509: 575-581Crossref PubMed Scopus (1241) Google Scholar, 4Wilhelm M. Schlegl J. Hahne H. Moghaddas Gholami A. Lieberenz M. Savitski M.M. Ziegler E. Butzmann L. Gessulat S. Marx H. Mathieson T. Lemeer S. Schnatbaum K. Reimer U. Wenschuh H. et al.Mass-spectrometry-based draft of the human proteome.Nature. 2014; 509: 582-587Crossref PubMed Scopus (1115) Google Scholar). Although providing a necessary foundation, these catalogs remain insufficient for describing the complex biological mechanisms at work within cells. At its heart, biology is the study of dynamic processes in living organisms, and proteins are the operators that exert direct control over processes at the cellular level. Today, we seek to build upon these genomic and proteomic foundations to understand closely related biological states, including the stages of a therapeutic response, cellular differentiation, and cancer progression. Such studies promise to reveal the core of mechanistic cell biology by elucidating the relationships between proteins and their roles in cellular processes. In the past half-century, key insights into many cellular processes have been revealed by demonstrating differential abundance of individual proteins across a small number of conditions. But as with a single photograph, information about a single protein in isolation provides only a narrow portal for viewing the dynamics of the cellular landscape. We now appreciate that the coordination of many proteins and the responses of multiprotein networks to the cellular environment define this landscape. To understand cellular mechanisms, experimental data showing the dynamic nature of the proteome under physiologic and experimentally manipulated conditions are required. In practice, this involves populating a multidimensional matrix of data “photographs” as either a function of time or of comparisons across many closely related states. Over the last 50 years, our understanding of the levels, localization, interactions, and activation states for proteins has exploded alongside the emergence of increasingly complex biochemical, biophysical, and molecular tools. Biochemical assays using radioisotopes (5Yalow R.S. Berson S.A. Immunoassay of endogenous plasma insulin in man.J. Clin. Invest. 1960; 39: 1157-1175Crossref PubMed Google Scholar, 6Walsh D.A. Perkins J.P. Krebs E.G. An adenosine 3′,5′-monophosphate-dependent protein kinase from rabbit skeletal muscle.J. Biol. Chem. 1968; 243: 3763-3765Abstract Full Text PDF PubMed Google Scholar) and spectrophotometric readouts (7Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) established a quantitative framework for understanding proteins and their functions, either individually or in small groups. With the emergence of monoclonal antibodies, molecular and cell biologists have gained the ability to probe any protein or any protein feature against which a specific reagent could be generated (8Köhler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.Nature. 1975; 256: 495-497Crossref PubMed Google Scholar). A parallel explosion in molecular biology made it possible to overexpress or knock down genes, append affinity tags (9Munro S. Pelham H.R. Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70.EMBO J. 1984; 3: 3087-3093Crossref PubMed Scopus (85) Google Scholar), and attach fluorescent reporters (10Tsien R.Y. The green fluorescent protein.Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4600) Google Scholar) to proteins in both cultured cells and in vivo. Additional techniques such as two-dimensional gel electrophoresis (11O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) allowed for the separation of complex protein mixtures. The most recent revolution in protein chemistry has come from the field of proteomic mass spectrometry (12Hunt D.F. Yates 3rd., J.R. Shabanowitz J. Winston S. Hauer C.R. Protein sequencing by tandem mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 6233-6237Crossref PubMed Scopus (1043) Google Scholar), opening the door to the direct characterization of proteins and post-translational modifications (PTMs) 1The abbreviations used are:PTMpost-translational modificationiTRAQisobaric tags for relative and absolute quantificationTMTtandem mass tagSILACstable isotope-labeling of amino acids in cell culture. with site-specific resolution. post-translational modification isobaric tags for relative and absolute quantification tandem mass tag stable isotope-labeling of amino acids in cell culture. Mass spectrometry (MS) provides access to the proteome through three main avenues: identifying the proteins present, assessing their post-translational modification states, and quantifying the relative abundance of each protein-modification state combination (13Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5183) Google Scholar, 14Ong S.-E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat. Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1251) Google Scholar, 15Domon B. Aebersold R. Mass spectrometry and protein analysis.Science. 2006; 312: 212-217Crossref PubMed Scopus (1463) Google Scholar, 16Zhang Y. Fonslow B.R. Shan B. Baek M.-C. Yates 3rd, J.R. Protein analysis by shotgun/bottom-up proteomics.Chem. Rev. 2013; 113: 2343-2394Crossref PubMed Scopus (696) Google Scholar, 17Grimsrud P.A. Swaney D.L. Wenger C.D. Beauchene N.A. Coon J.J. Phosphoproteomics for the masses.ACS Chem. Biol. 2010; 5: 105-119Crossref PubMed Scopus (137) Google Scholar). Although characterization of protein-modification state combinations would ideally be done on intact proteins (18Tran J.C. Zamdborg L. Ahlf D.R. Lee J.E. Catherman A.D. Durbin K.R. Tipton J.D. Vellaichamy A. Kellie J.F. Li M. Wu C. Sweet S.M. Early B.P. Siuti N. LeDuc R.D. Compton P.D. Thomas P.M. Kelleher N.L. Mapping intact protein isoforms in discovery mode using top-down proteomics.Nature. 2011; 480: 254-258Crossref PubMed Scopus (443) Google Scholar) to reveal the full repertoire of “proteoforms” (19Smith L.M. Kelleher N.L. Consortium for Top Down Proteomics Proteoform: a single term describing protein complexity.Nat. Methods. 2013; 10: 186-187Crossref PubMed Scopus (653) Google Scholar), the majority of “proteomic” analyses are actually performed on peptides generated by proteolytic digestion of protein samples (12Hunt D.F. Yates 3rd., J.R. Shabanowitz J. Winston S. Hauer C.R. Protein sequencing by tandem mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 6233-6237Crossref PubMed Scopus (1043) Google Scholar). With a focus on speed, sensitivity, and dynamic range in sequencing peptides from complex mixtures, improvements in mass spectrometry instrumentation have brought about dramatic improvements in cataloging the protein constituents of a sample; recent reports have shown the ability to identify the entire yeast proteome in an hour (20Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The one hour yeast proteome.Mol. Cell. Proteomics. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar) and provided a draft of the human proteome (3Kim M.-S. Pinto S.M. Getnet D. Nirujogi R.S. Manda S.S. Chaerkady R. Madugundu A.K. Kelkar D.S. Isserlin R. Jain S. Thomas J.K. Muthusamy B. Leal-Rojas P. Kumar P. Sahasrabuddhe N.A. et al.A draft map of the human proteome.Nature. 2014; 509: 575-581Crossref PubMed Scopus (1241) Google Scholar, 4Wilhelm M. Schlegl J. Hahne H. Moghaddas Gholami A. Lieberenz M. Savitski M.M. Ziegler E. Butzmann L. Gessulat S. Marx H. Mathieson T. Lemeer S. Schnatbaum K. Reimer U. Wenschuh H. et al.Mass-spectrometry-based draft of the human proteome.Nature. 2014; 509: 582-587Crossref PubMed Scopus (1115) Google Scholar). Likewise, sophisticated metal- and immunoaffinity-based enrichment methods (21Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1431) Google Scholar, 22Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.-M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (907) Google Scholar) have proven useful in elucidating the repertoire of proteins carrying PTMs (23Olsen J.V. Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Focused examples involving phosphorylation (17Grimsrud P.A. Swaney D.L. Wenger C.D. Beauchene N.A. Coon J.J. Phosphoproteomics for the masses.ACS Chem. Biol. 2010; 5: 105-119Crossref PubMed Scopus (137) Google Scholar, 24Macek B. Mann M. Olsen J.V. Global and site-specific quantitative phosphoproteomics: principles and applications.Annu. Rev. Pharmacol. Toxicol. 2009; 49: 199-221Crossref PubMed Scopus (328) Google Scholar), ubiquitination (25Bustos D. Bakalarski C.E. Yang Y. Peng J. Kirkpatrick D.S. Characterizing ubiquitination sites by peptide-based immunoaffinity enrichment.Mol. Cell. Proteomics. 2012; 11: 1529-1540Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 26Ordureau A. Münch C. Harper J.W. Quantifying ubiquitin signaling.Mol. 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Obtaining abundance measurements for proteins and their various modification states across conditions produces a detailed picture of protein activity and is now possible via a myriad of technologies. In the simplest form of quantitative mass spectrometry, label-free analysis determines the signal intensities or peak areas associated with individual peptides (30Neilson K.A. Ali N.A. Muralidharan S. Mirzaei M. Mariani M. Assadourian G. Lee A. van Sluyter S.C. Haynes P.A. Less label, more free: approaches in label-free quantitative mass spectrometry.Proteomics. 2011; 11: 535-553Crossref PubMed Scopus (473) Google Scholar). Binary and ternary comparisons provide additional accuracy over label-free techniques and have become routine using methods such as stable isotope-labeling of amino acids in cell culture (SILAC) (31Ong S.-E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4222) Google Scholar) and reductive methylation (32Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protoc. 2009; 4: 484-494Crossref PubMed Scopus (929) Google Scholar), as have comparisons of 4–10 conditions using isobaric tags for relative and absolute quantification (iTRAQ) (33Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. et al.Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. 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Quantitative mass spectrometry in proteomics: critical review update from 2007 to the present.Anal. Bioanal. Chem. 2012; 404: 939-965Crossref PubMed Scopus (519) Google Scholar, 37Liebler D.C. Zimmerman L.J. Targeted quantitation of proteins by mass spectrometry.Biochemistry. 2013; 52: 3797-3806Crossref PubMed Scopus (196) Google Scholar, 38Law K.P. Lim Y.P. Recent advances in mass spectrometry: data independent analysis and hyper reaction monitoring.Expert Rev. Proteomics. 2013; 10: 551-566Crossref PubMed Scopus (94) Google Scholar, 39Chapman J.D. Goodlett D.R. Masselon C.D. Multiplexed and data-independent tandem mass spectrometry for global proteome profiling.Mass Spectrom. Rev. 2014; 33: 452-470Crossref PubMed Scopus (144) Google Scholar). Finally, although a full description of such methods falls outside the scope of this review, significant advances have been made in data-independent analysis (40Venable J.D. Dong M.-Q. Wohlschlegel J. Dillin A. Yates J.R. Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra.Nat. Methods. 2004; 1: 39-45Crossref PubMed Scopus (505) Google Scholar). These methods include both hypothesis-driven, high-throughput targeted analyses by selected and multiple reaction monitoring (37Liebler D.C. Zimmerman L.J. Targeted quantitation of proteins by mass spectrometry.Biochemistry. 2013; 52: 3797-3806Crossref PubMed Scopus (196) Google Scholar, 41Anderson L. Hunter C.L. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.Mol. Cell. Proteomics. 2006; 5: 573-588Abstract Full Text Full Text PDF PubMed Scopus (1041) Google Scholar, 42Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2007; 6: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 43Lange V. 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Targeted proteomic quantification on quadrupole-orbitrap mass spectrometer.Mol. Cell. Proteomics. 2012; 11: 1709-1723Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Such methods can be leveraged to generate proteome maps through targeted post hoc data extraction (47Gillet L.C. Navarro P. Tate S. Röst H. Selevsek N. Reiter L. Bonner R. Aebersold R. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis.Mol. Cell. Proteomics. 2012; 11 (O111.016717)Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar), providing a complement to conventional data-dependent analyses. Proteomics technologies have reached the stage where studies comparing many samples are now feasible, facilitating time course experiments, multiple condition comparisons, and facile introduction of biological replicates. Each quantitative MS technique has its own strengths and weaknesses, and the pairing of an experiment to the right quantitative model is essential to maximize the utility of the results. A biologist should consider the following questions. What are the key points in designing a successful proteomics analysis? How does one choose among the available proteomics technologies? How and when is multiplexing valuable? Here, we provide background on quantitative proteomic techniques useful for the parallel analysis of multiple samples, and we discuss their benefits and limitations in addressing biological questions. Many discovery proteomics experiments today utilize data-dependent tandem mass spectrometry. In data-dependent analysis, the instrument is programmed to first generate an MS1 spectrum that surveys the masses and signal intensities of intact peptide ions. As this information is frequently insufficient to conclusively match spectra to peptides, the instrument performs a series of secondary MS scans (termed MS/MS or MS2), where individual peptide ions are isolated and fragmented along their amide backbones. Differences in mass between these peptide fragment ions are used to decipher peptide sequence information (48Steen H. Mann M. The ABC's (and XYZ's) of peptide sequencing.Nat. Rev. Mol. Cell Biol. 2004; 5: 699-711Crossref PubMed Scopus (769) Google Scholar). Although the intensities of ions observed in an MS1 scan are generally proportional to peptide abundance in the sample, absolute signal intensities can vary depending on a number of factors. Run-to-run differences in sample complexity, chromatography, data-dependent sampling, and peptide ionization efficiency have historically limited the reliability of comparing peptide ion signals across runs. Even with biological replicates in hand, the prevalence of missing values in proteomic data has posed a challenge for downstream statistical analysis. A breakthrough for quantitative proteomics came in the application of stable isotope dilution approaches to the quantitation of proteins and digested peptides. Stable isotopes such as 13C, 15N, 18O, and 2H (deuterium) can be introduced to proteomic samples in a variety of ways (Fig. 1). An early approach, isotope-coded affinity tags (ICAT), employs a biotin affinity tag coupled to a stable isotope-labeled (i.e. 8 deuterium; 2H8) linker and a thiol-reactive group (49Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4180) Google Scholar). These tags allow for quantitation of cysteine-containing peptides via MS1 survey scans where intensities of peptide ions labeled with the “light” and “heavy” ICAT reagents are directly compared. This approach provides relative ratio measurements of peptides between samples as a proxy for overall proteoform abundance. Although effective, the reliance on cysteine-containing peptides means the majority of peptides lacking cysteine are unmeasured. Thus, alternative techniques such as dimethyl labeling, quantitative carbamylation, and the incorporation of other stable isotopes (18O and 15N) were subsequently developed (50Hsu J.-L. Huang S.-Y. Chow N.-H. Chen S.-H. Stable-isotope dimethyl labeling for quantitative proteomics.Anal. Chem. 2003; 75: 6843-6852Crossref PubMed Scopus (547) Google Scholar, 51Boersema P.J. Aye T.T. van Veen T.A. Heck A.J. Mohammed S. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates.Proteomics. 2008; 8: 4624-4632Crossref PubMed Scopus (174) Google Scholar, 52Angel P.M. Orlando R. Quantitative carbamylation as a stable isotopic labeling method for comparative proteomics.Rapid Commun. Mass Spectrom. 2007; 21: 1623-1634Crossref PubMed Scopus (17) Google Scholar, 53Murphy R.C. Clay K.L. Synthesis and back exchange of 18O labeled amino acids for use as internal standards with mass spectrometry.Biomed. Mass Spectrom. 1979; 6: 309-314Crossref PubMed Google Scholar, 54Conrads T.P. Alving K. Veenstra T.D. Belov M.E. Anderson G.A. Anderson D.J. Lipton M.S. Pasa-Tolić L. Udseth H.R. Chrisler W.B. Thrall B.D. Smith R.D. Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.Anal. Chem. 2001; 73: 2132-2139Crossref PubMed Scopus (258) Google Scholar). An inherent challenge with chemical labeling approaches is that they only account for differences in sample preparation that occur after the labeling step. Seeking to minimize the variability imparted through sample handling, metabolic labeling was developed for proteomics (55Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Accurate quantitation of protein expression and site-specific phosphorylation.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 6591-6596Crossref PubMed Scopus (922) Google Scholar). A popular application of metabolic labeling is SILAC (31Ong S.-E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4222) Google Scholar), where one or more naturally occurring amino acids are replaced with synthetic counterparts, enriched in the stable isotopes 13C and 15N. In common practice, fully labeled [13C615N2]lysine and [13C615N4]arginine are used in combination so that all peptides arising from trypsin digestion (except for those at the C terminus of the protein) can be systematically quantified. Incorporating 13C and 15N labels alleviates the chromatographic retention time shifts sometimes observed with 2H labeling. Amino acid reagents such as [13C6]lysine, [13C6]arginine, and [15N4]arginine (with lesser numbers of stable isotopes incorporated) are also useful and can be combined with fully labeled reagents to extend multiplexing beyond simple paired comparisons. In fact, proof-of-concept studies examining adipocyte differentiation and tyrosine phosphorylation dynamics have demonstrated 5-plex quantitation using these and additional forms of arginine (i.e. 13C615N42H7) (56Molina H. Yang Y. Ruch T. Kim J.-W. Mortensen P. Otto T. Nalli A. Tang Q.-Q. Lane M.D. Chaerkady R. Pandey A. Temporal profiling of the adipocyte proteome during differentiation using a five-plex SILAC based strategy.J. Proteome Res. 2009; 8: 48-58Crossref PubMed Scopus (109) Google Scholar) or combinations of labeled lysine, arginine, and tyrosine (57Tzouros M. Golling S. Avila D. Lamerz J. Berrera M. Ebeling M. Langen H. Augustin A. Development of a 5-plex SILAC method tuned for the quantitation of tyrosine phosphorylation dynamics.Mol. Cell. Proteomics. 2013; 12: 3339-3349Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), respectively. Nevertheless, a limiting factor of multiplexing capacity in traditional SILAC has been the overlap in the mass dimension between different isotopically labeled forms, even in high resolution MS1 spectra. Moreover, splitting the signal across labeled forms of the same peptide increases the complexity of signals seen by the mass spectrometer, thereby impacting sensitivity and peptide identification rate. One solution for multiplexing is to use standard metabolic labeling to “unroll” a large sample set into a series of binary comparisons. Instead of labeling multiple samples, this approach involves the preparation of an isotopically labeled reference standard that can be added to each experimental contrast. Comparing each sample against a single common reference mixture makes it possible to determine relative peptide abundance as a ratio of ratios. An advantage of this over label-free analysis is that the presence of reference features in each biological sample empowers longitudinal studies by permitting MS1-based quantification even when the corresponding features are absent (58Geiger T. Cox J. Ostasiewicz P. Wisniewski J.R. Mann M. Super-SILAC mix for quantitative proteomics of human tumor tissue.Nat. Methods. 2010; 7: 383-385Crossref PubMed Scopus (402) Google Scholar, 59Kirkpatrick D.S. Bustos D.J. Dogan T. Chan J. Phu L. Young A. Friedman L.S. Belvin M. Song Q. Bakalarski C.E. Hoeflich K.P. Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 19426-19431Crossref PubMed Scopus (37) Google Scholar, 60Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (1874) Google Scholar). This approach is equally applicable in the context of both data-independent analysis and targeted experiments, where individual samples are compared with a common reference sample. A variety of approaches for the construction of a common reference sample has been reported, ranging from focused studies using isotopically labeled synthetic peptides (i.e. AQUA) (61Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1464) Google Scholar) or recombinant proteins (62Hanke S. Besir H. Oesterhelt D. Mann M. Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level.J. Proteome Res. 2008; 7: 1118-1130Crossref PubMed Scopus (176) Google Scholar) to more global studies employing labeled cell lysates (63Ishihama Y. Sato T. Tabata T. Miyamoto N. Sagane K. Nagasu T. Oda Y.

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.

Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.

DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.

Advances in high resolution tandem mass spectrometry and peptide enrichment technologies have transformed the field of protein biochemistry by enabling analysis of end points that have traditionally been inaccessible to molecular and biochemical techniques. One field benefitting from this research has been the study of ubiquitin, a 76-amino acid protein that functions as a covalent modifier of other proteins. Seminal work performed decades ago revealed that trypsin digestion of a branched protein structure known as A24 yielded an enigmatic diglycine signature bound to a lysine residue in histone 2A. With the onset of mass spectrometry proteomics, identification of K-GG-modified peptides has emerged as an effective way to map the position of ubiquitin modifications on a protein of interest and to quantify the extent of substrate ubiquitination. The initial identification of K-GG peptides by mass spectrometry initiated a flurry of work aimed at enriching these post-translationally modified peptides for identification and quantification en masse. Recently, immunoaffinity reagents have been reported that are capable of capturing K-GG peptides from ubiquitin and its thousands of cellular substrates. Here we focus on the history of K-GG peptides, their identification by mass spectrometry, and the utility of immunoaffinity reagents for studying the mechanisms of cellular regulation by ubiquitin.

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin’s conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin–deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.

Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941-mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo.

Arsenic is a known human carcinogen that affects a variety of processes within the cell. In this study, the effects of environmentally relevant As(III) exposures on the ubiquitin (Ub)-proteasome pathway have been investigated. Low-level As(III) exposure (0.5 - 10 microM) causes an accumulation of high-molecular-weight ubiquitin protein conjugates in both precision-cut rabbit renal-cortical slices and human embryonic kidney (HEK) 293 cells. The As(III) doses that induced these molecular changes were subcytotoxic in both model systems. Doses of 10 microM As(III) decreased cellular activity of the 20S proteasome by 40 and 15% in slices and HEK293 cells, respectively. As(III) did not cause any notable difference in Ub-conjugating activity of rabbit renal slices or HEK293 cells. Since ubiquitination plays such a vital role in maintaining cellular homeostasis, this noticeable perturbation of cellular ubiquitination is likely to have a multitude of signaling effects within the cells and may contribute to the pathogenesis of low-level arsenic.

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006 Genomics 88, 731-736). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans. Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues (Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006 Genomics 88, 731-736). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans. Hominoid- or human-specific genes are among the most important resources to have emerged from the completion of the human genome project. Still, we know little about this small group of genes, which presumably modulate or regulate signaling pathways that have evolved to a higher level of complexity and that distinguish hominoids and humans from less complex species. TBC1D3 belongs to a hominoid-specific gene family with no known orthologs outside of the primate lineage (1Hodzic D. Kong C. Wainszelbaum M.J. Charron A.J. Su X. Stahl P.D. Genomics. 2006; 88: 731-736Crossref PubMed Scopus (46) Google Scholar). TBC1D3 was originally identified by Pei et al. as a prostate and breast cancer oncogene (2Pei L. Peng Y. Yang Y. Ling X.B. Van Eyndhoven W.G. Nguyen K.C. Rubin M. Hoey T. Powers S. Li J. Cancer Res. 2002; 62: 5420-5424PubMed Google Scholar). The TBC1D3 genes are arrayed along a region of human chromosome 17 that has undergone extensive intrachromosomal rearrangement and segmental duplication following its probable recent appearance within the hominoid lineage (3Zody M.C. Garber M. Adams D.J. Sharpe T. Harrow J. Lupski J.R. Nicholson C. Searle S.M. Wilming L. Young S.K. Abouelleil A. Allen N.R. Bi W. Bloom T. Borowsky M.L. Bugalter B.E. Butler J. Chang J.L. Chen C.K. Cook A. Corum B. Cuomo C.A. de Jong P.J. DeCaprio D. Dewar K. FitzGerald M. Gilbert J. Gibson R. Gnerre S. Goldstein S. Grafham D.V. Grocock R. Hafez N. Hagopian D.S. Hart E. Norman C.H. Humphray S. Jaffe D.B. Jones M. Kamal M. Khodiyar V.K. LaButti K. Laird G. Lehoczky J. Liu X. Lokyitsang T. Loveland J. Lui A. Macdonald P. Major J.E. Matthews L. Mauceli E. McCarroll S.A. Mihalev A.H. Mudge J. Nguyen C. Nicol R. O'Leary S.B. Osoegawa K. Schwartz D.C. Shaw-Smith C. Stankiewicz P. Steward C. Swarbreck D. Venkataraman V. Whittaker C.A. Yang X. Zimmer A.R. Bradley A. Hubbard T. Birren B.W. Rogers J. Lander E.S. Nusbaum C. Nature. 2006; 440: 1045-1049Crossref PubMed Scopus (110) Google Scholar). Chromosome 17 has also been implicated in a wide variety of human genetic diseases and encodes genes involved in breast cancer (BRCA1), neurofibromatosis (NFI), and the DNA damage response (TP53). TBC1D3 (also known as PRC17), which encodes a protein containing a TBC (Tre-2, BUB2, cdc16) domain, was shown to induce tumors in nude mice and growth in low serum when exogenously expressed in mouse 3T3 fibroblasts (2Pei L. Peng Y. Yang Y. Ling X.B. Van Eyndhoven W.G. Nguyen K.C. Rubin M. Hoey T. Powers S. Li J. Cancer Res. 2002; 62: 5420-5424PubMed Google Scholar). Previous work from our laboratory identified eight highly related TBC1D3 paralogs, organized in two clusters within the 17q12 genomic region. These genes potentially encode six individual TBC1D3 variants with differences in a handful of amino acids within the TBC domain. Interestingly, we reported a tissue-specific transcription pattern of TBC1D3 paralogs among normal human tissues and documented alterations in this pattern in several prostate tumors (1Hodzic D. Kong C. Wainszelbaum M.J. Charron A.J. Su X. Stahl P.D. Genomics. 2006; 88: 731-736Crossref PubMed Scopus (46) Google Scholar).The EGF 5The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; siRNA, small interference RNA; RT, reverse transcription; PBS, phosphate-buffered saline; RBD, Ras binding domain; GST, glutathione S-transferase; CMV, cytomegalovirus; E3, ubiquitin-protein isopeptide ligase; EEA1, early endosomal antigen 1. 5The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; siRNA, small interference RNA; RT, reverse transcription; PBS, phosphate-buffered saline; RBD, Ras binding domain; GST, glutathione S-transferase; CMV, cytomegalovirus; E3, ubiquitin-protein isopeptide ligase; EEA1, early endosomal antigen 1. receptor (EGFR) is among the best studied receptor tyrosine kinases. Once EGFR is activated by its ligand, receptor dimerization and autophosphorylation occur (4Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar), followed by the recruitment of multiple adaptor proteins. Activated EGFRs are ubiquitinated, internalized, and transported along the endocytic pathway while maintaining signaling. EGFR belongs to the ERBB receptor signal transduction network, extensively implicated in human cancer and a target for cancer therapeutics (5Citri A. Yarden Y. Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1578) Google Scholar). Numerous aberrations have been shown in ERBB receptor genes (deletions, insertions, and point mutations) that may alter both signaling and membrane trafficking pathways (6Lynch T.J. Bell D.W. Sordella R. Gurubhagavatula S. Okimoto R.A. Brannigan B.W. Harris P.L. Haserlat S.M. Supko J.G. Haluska F.G. Louis D.N. Christiani D.C. Settleman J. Haber D.A. N. Engl. J. Med. 2004; 350: 2129-2139Crossref PubMed Scopus (9913) Google Scholar, 7Paez J.G. Janne P.A. Lee J.C. Tracy S. Greulich H. Gabriel S. Herman P. Kaye F.J. Lindeman N. Boggon T.J. Naoki K. Sasaki H. Fujii Y. Eck M.J. Sellers W.R. Johnson B.E. Meyerson M. Science. 2004; 304: 1497-1500Crossref PubMed Scopus (8398) Google Scholar). Normal tissue morphogenesis, homeostasis, and growth depend on finely orchestrated cellular responses to growth factors and cytokines. Thus, it has been proposed that dysregulation of the endocytic machinery might lead to uncontrolled cell growth by disturbing the specificity and duration of one or more signaling pathways (8Crosetto N. Tikkanen R. Dikic I. FEBS Lett. 2005; 579: 3231-3238Crossref PubMed Scopus (15) Google Scholar).We speculated that TBC1D3 might modulate EGFR signaling and trafficking events triggering cell proliferation. In the present work, we examined the signaling, trafficking, and fate of the EGFR in murine and human cells expressing or depleted of TBC1D3. We show that the activation of Erk and protein kinase B/Akt pathways, the kinetics of receptor trafficking, and EGF-induced cell proliferation are modulated by TBC1D3 expression. Understanding the mode and mechanism of action of TBC1D3 may provide a unique insight in the “biological rationale” by which recently evolved genes modulate signaling pathways in a human-specific manner and thereby create models for exploring the role of human-specific genes in human metabolic control and physiology.EXPERIMENTAL PROCEDURESReagents—125I-Labeled human EGF and [methyl-3H]thymidine were purchased from Amersham Biosciences. Antibodies against phospho-Akt, phospho-MAPK, total Akt, and total MAPK were from Cell Signaling Technology (Beverly, MA). Cbl antibody was from BD Transduction Laboratories (Rockville, MD). Monoclonal EGFR (Ab-5) antibody was purchased from Calbiochem (San Diego, CA). Monoclonal Ras and polyclonal EGFR antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal α-tubulin was purchased from Sigma-Aldrich. The ubiquitin antibody was from Zymed Laboratories Inc. (San Francisco, CA). The monoclonal antibody directed against the C-terminal 50 amino acids of TBC1D3 was generated by the Hybridoma Center at Washington University (St. Louis, MO). Alexa-Fluor conjugated antibodies were from Molecular Probes (Carlsbad, CA).Construction of Recombinant Retroviruses and Stable Cell Lines Expressing TBC1D3—TBC1D3 paralog D cDNA (HGCN designation), kindly provided by Dr. Jing Li (Tularik Inc, San Francisco, CA), was subcloned into EcoRI/AccI restriction sites of the pBABE-puro vector using the forward primer 5′-CACCATGGACGTGGTAGAGGTCG-3′ and the reverse primer 5′-CTAGAAGCCTGGAGGGAACTG-3′. To make stable cell lines, 293T-packaging cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin. When cells reached 80% confluence, they were transfected with either pBABE-puro:TBC1D3 or empty pBABE-puro along with pCLEco (replication-incompetent helper plasmid) for mouse cells or pUMVC3 plus pCVM-VSV-G for human cells. Retroviral supernatant was collected after 48 h and used with Polybrene (5 μg/ml) to infect either human DU145 prostate cancer cells or mouse NR6 fibroblasts already stably expressing the human EGFR (NR6:hEGFR) (9Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar). After incubation for 48 h the infected cells were selected with medium containing puromycin (2 μg/ml). Single clones were grown to obtain clonal stable cell lines.TBC1D3 Silencing—TBC1D3 was successfully knocked down using a commercial siRNA pool (SMARTpool, Dharmacon). Briefly, DU145 cells were transfected with TBC1D3 siRNA (50 nm final concentration) or irrelevant siRNA (scramble siRNA, Ambion) using Lipofectamine 2000 and were used 36 h later for the appropriate experiments. TBC1D3 depletion was evaluated by RT-PCR. Cells were subjected to RNA extraction in RNase-free conditions using TRIzol (Invitrogen). The RNA was then used to detect TBC1D3 transcript levels by RT-PCR.Cell Proliferation Assay—Cell growth was measured by thymidine incorporation into DNA. The cells (1 × 105 cells/well) were serum-starved and incubated in the presence or absence of 100 ng/ml EGF for 36 h. 1 μCi/ml [methyl-3H]thymidine (2 Ci/mmol) was added in the last 6 h. The cells were then washed three times with phosphate-buffered saline (PBS). The incorporation of [3H]thymidine into DNA was determined after DNA precipitation with cold 10% (w/v) trichloroacetic acid and cell solubilization in 0.1 m NaOH. Tritium was measured by scintillation counting.Ras Activation Assay—Raf-1 RBD (Ras binding domain) expressed as a GST fusion protein was purified from Escherichia coli by immobilization on glutathione-Sepharose beads and used to affinity precipitate the active form of Ras (Ras-GTP) from cell extracts (10Taylor S.J. Resnick R.J. Shalloway D. Methods Enzymol. 2001; 333: 333-342Crossref PubMed Scopus (72) Google Scholar). Active Ras precipitation was visualized by SDS-PAGE and Western blotting with a monoclonal pan-Ras antibody.Lysate Preparation, SDS-PAGE, and Western Blotting—To prepare whole cell lysates, cell monolayers were washed with PBS and lysed in ice-cold lysis buffer (PBS, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). The lysates were clarified by centrifugation, and protein concentrations were determined using the BCA Protein Assay Reagent Kit (Pierce). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes, which were blocked and probed with the indicated antibodies. To determine relative protein amounts, three representative exposures for each sample were quantified using AlphaEaseFc software (Alpha Innotech Corp., San Leandro, CA).Signaling and EGFR Degradation Assays—Akt and Erk1/2 activation and EGFR degradation were measured in cells serum-starved for 5 h. Cells were incubated in the presence of EGF (100 ng/ml) at 37 °C for different time points, washed with ice-cold PBS, and lysed as described above. Proteins were separated by SDS-PAGE and analyzed by Western blotting with phospho-Akt, phospho-Erk1/2, and EGFR antibodies.Receptor Internalization—Cells expressing TBC1D3 or vector alone were serum-starved and then incubated at 4 °C for 1 h with 70 ng/ml 125I-EGF (750 Ci/mmol), washed with ice-cold PBS and warmed for different periods of time. At each time point, unbound ligand was removed by washing the monolayer five times with ice-cold PBS. Surface-bound and internalized ligands were measured as described previously (11Sorkin A. Waters C. Overholser K.A. Carpenter G. J. Biol. Chem. 1991; 266: 8355-8362Abstract Full Text PDF PubMed Google Scholar). Briefly, surface-bound ligand was collected in ice-cold “acid strip” buffer (50 mm glycine-HCl, 100 mm NaCl, 1 mg/ml polyvinylpyrrolidone, pH 3.0), and internalized ligands were released in 0.1 n NaOH. 125I-EGF was quantified by scintillation counting. Nonspecific binding (∼3%) was tested in the presence of unlabeled human EGF (200 nm, Sigma) and subtracted from the total. EGF internalization rates were estimated as the ratio of internalized to surface-bound EGF.EGFR Turnover—Cells were starved with media lacking cysteine and methionine plus 5% of dialyzed fetal bovine serum for 30 min at 37 °C and were pulse-labeled by incubation with 100 μCi/ml [35S]methionine (1175 Ci/mmol) in the same medium for 1 h. Cells were washed and incubated in cold medium supplemented with 2 mm each cysteine and methionine for the indicated times in the incubator. Thereafter cells were lysed in radioimmune precipitation assay buffer; EGFR was immunoprecipitated by incubation with monoclonal anti-EGFR antibody and resolved by SDS-PAGE. The gel was dried, and radioactivity was detected by autoradiography.Immunoprecipitation—All immunoprecipitations were performed by lysing the cells in ice-cold immunoprecipitation buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EGTA, 1 mm EDTA, 2.5 mm sodium pyrophosphate, 1 mm β-glycerolphosphate, 1 mm NaF, 1 mm sodium orthovanadate plus protease inhibitors). After clarification, protein concentrations were measured, and extracts were immunoprecipitated by incubation with the appropriate antibody followed by immobilization on Protein G-Sepharose beads (Sigma). Beads were resuspended in sample buffer and analyzed by SDS-PAGE and Western blot with the indicated antibodies.Liquid Chromatography-Tandem Mass Spectrometry Analysis of EGFR Ubiquitination—To examine the ubiquitination pattern of the EGFR in cells that express TBC1D3, the ubiquitin-AQUA method was used (12Kirkpatrick D.S. Hathaway N.A. Hanna J. Elsasser S. Rush J. Finley D. King R.W. Gygi S.P. Nat. Cell Biol. 2006; 8: 700-710Crossref PubMed Scopus (347) Google Scholar). Briefly, NR6:hEGFR: TBC1D3 and NR6:hEGFR:vector cells were grown in 150-mm dishes and treated or untreated with 100 ng/ml EGF for 5 min at 37 °C. Lysates were generated by the addition of TGH solubilization buffer (Triton X-100/glycerol/HEPES) containing 1% sodium deoxycholate and 10 mm N-ethylmaleimide. EGFR was immunoprecipitated by a monoclonal EGFR antibody. After sequential washes with TGH/sodium deoxycholate buffer containing 500 mm, 100 mm, and no NaCl, samples were separated by SDS-PAGE and analyzed by mass spectrometry using isotope-labeled internal standard peptides as previously described.Immunofluorescence—Cells were seeded onto glass coverslips (12 mm) at 0.5 × 105 per well, serum-starved, and cell-surface EGFR was saturated with 200 ng/ml EGF (Calbiochem) for 1 h at 4 °C. To follow internalization and trafficking of EGF, the cells were washed and placed in complete, pre-warmed (37 °C) media for different times. The coverslips were then washed with ice-cold PBS, and the cells were fixed for 20 min in PBS containing 3% (v/v) paraformaldehyde and autofluorescence quenched in 50 mm NH4Cl. The cells were permeabilized with PBS containing 0.05% (v/v) Triton X-100 for 10 min, and nonspecific binding sites were blocked with PBS containing 1% (w/v) bovine serum albumin and 2% goat serum. The coverslips were probed with primary antibodies followed by fluorophore-conjugated secondary antibodies and mounted onto slides with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA). Images were captured using a microscope equipped with a MRC1024 confocal LSM scanhead (Bio-Rad Laboratories).Statistical Analysis—All experiments presented were repeated a minimum of three times. The data represents the mean ± S.D. Student's t test was performed to calculate statistical significance.RESULTSTBC1D3 Increases Cell Growth and Enhances the Proliferative Response to EGF—Initial reports identifying TBC1D3 as an oncogene (2Pei L. Peng Y. Yang Y. Ling X.B. Van Eyndhoven W.G. Nguyen K.C. Rubin M. Hoey T. Powers S. Li J. Cancer Res. 2002; 62: 5420-5424PubMed Google Scholar) led us to investigate the effect of TBC1D3 expression and silencing on EGF-induced cell proliferation. The studies were carried out in DU145 cells, a well established human prostate carcinoma cell line. The cells were infected with retrovirus encoding TBC1D3D, and the resulting transductants were selected to obtain a clonal cell line expressing TBC1D3 (designated as DU145:TBC1D3). Similar results were obtained when different clonal cell lines were analyzed. DU145:vector (control) or DU145:TBC1D3 cells were incubated in the presence or absence of EGF for 36 h. The incorporation of [3H]thymidine into DNA was used as a measure of cell proliferation. The results (Fig. 1A) are expressed as the percentage of thymidine incorporation over control cells incubated without EGF (set at 100%). DU145:TBC1D3 cells grew more rapidly (∼2-fold), than control cells even in the absence of EGF. In the presence of EGF, both control and DU145:TBC1D3 cells displayed enhanced proliferation. However, EGF-treated DU145:TBC1D3 cells experienced a proliferative response that increased the amount of thymidine incorporation to ∼2.5-fold over that in vehicle-treated cells. Similar results were obtained using other human and mouse cell lines (data not shown). Experiments with murine lines correspond to a true null, because the gene is absent in the mouse genome. These results indicate that proliferation, the furthest downstream response to EGF, is augmented by the presence of TBC1D3, raising the question of how EGFR, TBC1D3, and proliferation are mechanistically linked.TBC1D3 Activates Ras and Enhances Ras Activation in Response to EGF—EGF receptor and other receptor tyrosine kinase are coupled to their downstream targets and to cell proliferation by Ras, which is constitutively activated in many human cancers (13Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). To determine whether Ras is activated in TBC1D3-expressing cells, GTP-Ras was monitored in cell lysates using the Raf-1 RBD pulldown assay (10Taylor S.J. Resnick R.J. Shalloway D. Methods Enzymol. 2001; 333: 333-342Crossref PubMed Scopus (72) Google Scholar). GTP-bound Ras was precipitated with GST-fused Raf-1 RBD from quiescent DU145 cells stably expressing TBC1D3 and control cells. GST alone was used as a control to estimate unspecific binding. The results showed that GTP-Ras was substantially increased (over 2-fold) in cells expressing TBC1D3 under steady-state conditions (Fig. 1B). To examine Ras following EGF stimulation, serum-starved TBC1D3 and control cells were stimulated for 10 min with 100 ng/ml EGF after which the lysates were incubated with GST or GST Raf1-RBD glutathione-Sepharose beads for 1 h. The Western blot in Fig. 1B demonstrates a robust increase in Ras activation in TBC1D3-expressing cells, which is enhanced almost 3-fold in response to EGF when compared with control cells. Similar activation profiles were obtained with other cell lines (data not shown).TBC1D3 and EGF Synergize in the Activation of the Erk1/2 and Akt Pathways—Signaling through Erk1/2 and protein kinase B/Akt are among the earliest post-EGF binding events associated with delayed apoptosis or cell proliferation. To investigate the effect of TBC1D3 on these two pathways, DU145 control or DU145:TBC1D3 cells were incubated with EGF at 37 °C, and levels of phospho-Erk1/2 and phospho-Akt were recorded by Western blotting. Total cellular contents of Erk1/2 or Akt were used to normalize the results in each sample. The data depicted in Fig. 2 show that following EGF stimulation both Erk1/2 and Akt activation were substantially elevated in cells expressing TBC1D3.FIGURE 2TBC1D3 expression enhances activation of Akt and Erk1/2 following EGF stimulation. DU145 cells expressing vector alone or TBC1D3 were serum-starved and incubated at 37 °C in the presence of 100 ng/ml EGF. The cells were washed, lysed, and analyzed by Western blot. The bar graphs were derived from densitometric analysis and show p-Erk1/2 and p-Akt normalized to the actual protein loaded.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A parallel study, measuring dose response to EGF, was carried out with a well established mouse NR6 fibroblast line that stably expresses both human EGFR (9Chen P. Gupta K. Wells A. J. Cell Biol. 1994; 124: 547-555Crossref PubMed Scopus (201) Google Scholar) and TBC1D3 (designated hereafter as NR6:hEGFR:TBC1D3). Murine cells do not express the hominoid-specific TBC1D3, and thus represent a null background, an optimal setting upon which to examine the effects of TBC1D3 expression. As shown in supplemental Fig. S1, control cells (vector alone) incubated with EGF, displayed activation kinetics that reached a maximum at 5 min for both Erk1/2 and Akt. NR6:hEGFR:TBC1D3 cells exhibited a heightened response to each concentration of EGF, also reaching a maximum at 5 min for both Erk1/2 and Akt. Additionally, Erk1/2 signaling in control cells subsided after 5 min, whereas TBC1D3-expressing cells sustained their Erk1/2 activation beyond 30 min. Thus, increases in both the magnitude and duration of Erk1/2 and Akt phosphorylation by TBC1D3 expression may account for the positive impact on cell proliferation observed following EGF stimulation.To rule out misleading phenotypes due to protein overexpression, we used RNA interference to suppress endogenous TBC1D3. DU145 cells were depleted of TBC1D3 using a specific commercial siRNA pool (Dharmacon). Transcript levels were evaluated by semi-quantitative RT-PCR 36 h later. A scrambled, irrelevant siRNA (Ambion) was used as a negative control. The extent of TBC1D3 silencing was ∼70% at the higher concentration assayed (Fig. 3A). Different concentrations of template demonstrated that the reactions fell within the linear range of template versus PCR product. In agreement with the results obtained when overexpressing TBC1D3, silencing TBC1D3 expression suppressed the activation of both Akt and Erk1/2 following EGF stimulation (Fig. 3B). Similar results were obtained with two additional in-house designed siRNAs (data not shown).FIGURE 3TBC1D3 depletion decreases Akt and Erk1/2 activation. A, semi-quantitative RT-PCR. TBC1D3 was suppressed in DU145 cells using a commercial siRNA pool (Dharmacon) as described under “Experimental Procedures.” Transcript levels of TBC1D3 (top panel) were determined by semi-quantitative RT-PCR from control cells (lanes 1 and 2), cells transfected with irrelevant siRNA (lanes 3 and 4) and cells transfected with TBC1D3 siRNA (lanes 5-10). B, DU145 cells were transfected with TBC1D3 siRNA (50 nm), irrelevant siRNA (irrel) or untransfected (Ctr). 36 h later the cells were serum-starved, incubated with EGF (100 ng/ml) at 37 °C for different time points, and analyzed by Western blot. The bar graphs were derived from densitometric analysis and show p-Erk1/2 and p-Akt normalized to the actual protein loaded.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EGF Binding and Internalization Are Enhanced by TBC1D3—EGF-EGFR internalization and degradation represent a well studied paradigm for receptor tyrosine kinase trafficking (14Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C.P. Walton G.M. Der C.J. Wiley H.S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 15Wells A. Welsh J.B. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 962-964Crossref PubMed Scopus (342) Google Scholar). To examine whether TBC1D3 regulates EGFR-mediated endocytosis, 125I-EGF uptake was measured in NR6:hEGFR: vector and NR6:hEGFR:TBC1D3 cells. Cells were serum-starved and then incubated with 125I-EGF (70 ng/ml) at 4 °C to allow ligand binding. After the cells were washed with ice-cold PBS, EGF uptake was evaluated by shifting the cells to 37 °C for different times. As shown in Fig. 4A, EGF accumulation, which plateaued at 15 min, was significantly greater in cells expressing TBC1D3. However, the rate of EGF internalization, expressed as the ratio of internalized to surface-bound EGF, was unaffected by the presence of TBC1D3 (inset in Fig. 4A). When cells were incubated with 125I-EGF for 3 h at 4 °C and surface-bound ligand was released, a 2-fold increase in EGF binding was recorded in cells expressing TBC1D3 (supplemental Fig. S2). Increased cell surface EGF binding and enhanced EGF internalization may be explained by higher levels of EGFR in cells that express TBC1D3. Indeed, TBC1D3-expressing cells contain ∼50% more EGFR at steady state than control cells (Fig. 4B). The data in Fig. 4B (right panel) show the densitometric analysis of multiple Western blots where EGFR signals were normalized to the total protein loaded. Similar results were obtained using human cell lines transiently transfected with TBC1D3 (data not shown).FIGURE 4TBC1D3 increases EGF internalization. A, NR6:hEGFR cells expressing TBC1D3 were serum-starved and incubated at 4 °C for 1 h with 125I-EGF (70 ng/ml). Internalization at 37 °C was quantified as described under “Experimental Procedures.” Inset: EGF internalization rate was estimated by the ratios of internalized to surface-bound radioactivity. B, EGFR levels are enhanced in cells expressing TBC1D3. NR6:hEGFR cells expressing TBC1D3 or vector alone were lysed and analyzed by Western blot. The bar graph (right panel) was derived from densitometric analysis and shows EGFR signals normalized to the actual protein loaded, using the α-tubulin signal (*, p < 0.01). The data are presented as means ± S.D. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EGFR Degradation Is Delayed by TBC1D3—Enhanced levels of EGFR in cells expressing TBC1D3 could be due to increased receptor synthesis, delayed receptor degradation, or both. Normally, EGF-EGFR binding results in the internalization and targeting of the receptor-ligand com

PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins. PIASy is a small ubiquitin-related modifier (SUMO) ligase that modifies chromosomal proteins in mitotic Xenopus egg extracts and plays an essential role in mitotic chromosome segregation. We have isolated a novel SUMO-2/3-modified mitotic chromosomal protein and identified it as poly(ADP-ribose) polymerase 1 (PARP1). PARP1 was robustly conjugated to SUMO-2/3 on mitotic chromosomes but not on interphase chromatin. PIASy promotes SUMOylation of PARP1 both in egg extracts and in vitro reconstituted SUMOylation assays. Through tandem mass spectrometry analysis of mitotically SUMOylated PARP1, we identified a residue within the BRCA1 C-terminal domain of PARP1 (lysine 482) as its primary SUMOylation site. Mutation of this residue significantly reduced PARP1 SUMOylation in egg extracts and enhanced the accumulation of species derived from modification of secondary lysine residues in assays using purified components. SUMOylation of PARP1 did not alter in vitro PARP1 enzyme activity, poly-ADP-ribosylation (PARylation), nor did inhibition of SUMOylation of PARP1 alter the accumulation of PARP1 on mitotic chromosomes, suggesting that SUMOylation regulates neither the intrinsic activity of PARP1 nor its localization. However, loss of SUMOylation increased PARP1-dependent PARylation on isolated chromosomes, indicating SUMOylation controls the capacity of PARP1 to modify other chromatin-associated proteins.

Abstract The order of addition of magnesium and glucose 1-phosphate to the phosphoenzyme (monophosphate phase) and of magnesium and glucose 1,6-diphosphate to the dephosphoenzyme (diphosphate phase) was investigated via substrate-velocity and equilibrium-isotope exchange experiments. All pathways ordered in both phases of the reaction were ruled out leaving only pathways random in one or both phases of the reaction; indicative evidence suggests that the pathway is actually random in both phases. The variations in maximum velocity and in the Michaelis constant for magnesium are described as a function of pH; the maximum velocity in the forward reaction is independent of pH from 6.5 to 8.3 while the Michaelis constant for magnesium depends on the basic form of two groups with pKa values in the range of 7.0 to 8.5.

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.

The dynamic and specific modification of cellular proteins by members of the ubiquitin protein family is a vital regulatory mechanism that lies at the heart of almost all biological processes. Because of both their pervasive and complex nature, these regulatory pathways have been the target of many recent proteomic studies. Such works have provided numerous insights. Through the use of various mass spectrometry techniques, affinity purification methods, and/or chemical probes, large lists have begun to be compiled for the multitude of substrates, interacting partners, and enzymatic components of these regulatory circuits. Furthermore, similar tools have provided many insights into functional aspects such as their mechanisms of substrate specificity and enzymatic activity. This review provides a summary of these recent proteomic works, along with comments on future directions of the field.

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.

A frameshift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin (Ub), UBB(+1). UBB(+1) has been considered to inhibit proteasomes and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrate that expression of extended Ub variants leads to accumulation of heterogeneously linked polyubiquitin conjugates, indicating a pervasive effect on Ub-dependent turnover. 20S proteasomes selectively proteolyzed Ub extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of Lys48 linkages at 26S proteasomes. Processing of Lys48 and Lys63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of Ub-dependent degradation by extended Ub variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.

In addition to amyloid-β plaques and tau tangles, mitochondrial dysfunction is implicated in the pathology of Alzheimer's disease (AD). Neurons heavily rely on mitochondrial function, and deficits in brain energy metabolism are detected early in AD; however, direct human genetic evidence for mitochondrial involvement in AD pathogenesis is limited. We analyzed whole-exome sequencing data of 4549 AD cases and 3332 age-matched controls and discovered that rare protein altering variants in the gene pentatricopeptide repeat-containing protein 1 (PTCD1) show a trend for enrichment in cases compared with controls. We show here that PTCD1 is required for normal mitochondrial rRNA levels, proper assembly of the mitochondrial ribosome and hence for mitochondrial translation and assembly of the electron transport chain. Loss of PTCD1 function impairs oxidative phosphorylation and forces cells to rely on glycolysis for energy production. Cells expressing the AD-linked variant of PTCD1 fail to sustain energy production under increased metabolic stress. In neurons, reduced PTCD1 expression leads to lower ATP levels and impacts spontaneous synaptic activity. Thus, our study uncovers a possible link between a protein required for mitochondrial function and energy metabolism and AD risk.SIGNIFICANCE STATEMENT Mitochondria are the main source of cellular energy and mitochondrial dysfunction is implicated in the pathology of Alzheimer's disease (AD) and other neurodegenerative disorders. Here, we identify a variant in the gene PTCD1 that is enriched in AD patients and demonstrate that PTCD1 is required for ATP generation through oxidative phosphorylation. PTCD1 regulates the level of 16S rRNA, the backbone of the mitoribosome, and is essential for mitochondrial translation and assembly of the electron transport chain. Cells expressing the AD-associated variant fail to maintain adequate ATP production during metabolic stress, and reduced PTCD1 activity disrupts neuronal energy homeostasis and dampens spontaneous transmission. Our work provides a mechanistic link between a protein required for mitochondrial function and genetic AD risk.

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

Dysregulation of mitophagy, whereby damaged mitochondria are labeled for degradation by the mitochondrial kinase PINK1 and E3 ubiquitin ligase Parkin with phosphorylated ubiquitin chains (p-S65 ubiquitin), may contribute to neurodegeneration in Parkinson’s disease. Here, we identify a phosphatase antagonistic to PINK1, protein phosphatase with EF-hand domain 2 (PPEF2), that can dephosphorylate ubiquitin and suppress PINK1-dependent mitophagy. Knockdown of PPEF2 amplifies the accumulation of p-S65 ubiquitin in cells and enhances baseline mitophagy in dissociated cortical cultures. Overexpressing enzymatically active PPEF2 reduces the p-S65 ubiquitin signal in cells, and partially purified PPEF2 can dephosphorylate recombinant p-S65 ubiquitin chains in vitro. Using a mass spectrometry approach, we have identified several p-S65-ubiquitinated proteins following mitochondrial damage that are inversely regulated by PPEF2 and PINK1. Interestingly, many of these proteins are involved in nuclear processes such as DNA repair. Collectively, PPEF2 functions to suppress mitochondrial quality control on a cellular level through dephosphorylation of p-S65 ubiquitin.

The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPhosphonoprotein. Characterization of aminophosphonic acid rich glycoproteins from sea anemonesDonald S. Kirkpatrick and Stephen H. BishopCite this: Biochemistry 1973, 12, 15, 2829–2840Publication Date (Print):July 1, 1973Publication History Published online1 May 2002Published inissue 1 July 1973https://doi.org/10.1021/bi00739a009RIGHTS & PERMISSIONSArticle Views74Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alertsclose Get e-Alerts

Treatment of (C6F5)2ECl (E = P or As) with the anions [π-C5H5Fe(CO)2]– and [π-C5H5Mo(CO)3]– affords the mononuclear complexes (C6F5)2EFe(CO)2π-C5H5 and (C6H5)2EMo(CO)3π-C5H5. The arsenic complexes can also be prepared by reaction of [(C6F5)2As]2 with [π-C5H5Fe(CO)2]2 and [π-C5H5Mo(CO)3]2. Attempts to capture (C6F5)2PLi with π-C5H5Fe(CO)2Cl or π-C5H5Mo(CO)3Cl were unsuccessful. U.v. irradiation of the mononuclear arsenic complexes gave [(C6F5)2AsFe(CO)π-C5H5]n and [(C6F5)2AsMO(CO)2π-C5H5]n. Reaction of [(C6F5)2E]2 with Fe3(CO)12 gave the bridged binuclear complexes [(C6F5)2PFe(CO)3]2 and [(C6F5)2AsFe(CO)3]2. The binuclear phosphorus complex is also obtained on heating (C6F5)2PH with Fe3(CO)12. Triruthenium dodecacarbonyl reacts with [(C6F5)2E]2 to give [(C6F5)2PRu(CO)3]2 or [(C6F5)2AsRu(CO)3]2.

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein’s structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques—the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.

Inositol 1,4,5-trisphosphate (IP₃) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP₃-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP₃ levels, IP₃ receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP₃ receptor (IP₃R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates. Our findings are (i) that at least 11 of the 167 lysines in IP₃R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least ∼40! of the IP₃R1-associated ubiquitin is monoubiquitin, (iii) that both Lys⁴⁸ and Lys⁶³ linkages are abundant in attached ubiquitin chains, and (iv) that Lys⁶³ linkages accumulate most rapidly. Additionally, we find that not all IP₃R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP₃R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP₃R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP₃R1 ubiquitination and degradation are highly complex processes.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Article21 December 2006free access The ubiquitin–proteasome system regulates membrane fusion of yeast vacuoles Maurits F Kleijnen Corresponding Author Maurits F Kleijnen Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Donald S Kirkpatrick Donald S Kirkpatrick Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Maurits F Kleijnen Corresponding Author Maurits F Kleijnen Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Donald S Kirkpatrick Donald S Kirkpatrick Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Maurits F Kleijnen 1, Donald S Kirkpatrick1 and Steven P Gygi1 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 432 1291; Fax: +1 617 432 1144; E-mail: [email protected] The EMBO Journal (2007)26:275-287https://doi.org/10.1038/sj.emboj.7601486 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin–proteasome regulation may be involved in membrane fusion elsewhere. Introduction The proteasome consists of a cylindrical core particle (CP)—a multi-protein complex that contains proteolytic active sites—and two regulatory particles (RP), one at each end of the cylinder. Its most important function is to recognize and degrade proteins that have been tagged with a multiubiquitin chain (Glickman and Ciechanover, 2002). Ubiquitination is a multi-stage process in which a succession of enzymes activate a single ubiquitin unit and then attach it either directly to a protein, or to an ubiquitin unit or chain that has already been attached to a protein (Glickman and Ciechanover, 2002). Thus, the ubiquitination process can result in a protein tagged with a single ubiquitin or, alternatively, a multiubiquitin chain. Proteins tagged with a single ubiquitin are not degraded by the proteasome and have important functions in endocytosis control and in the sorting of membrane proteins within late endosomes (also known as multivesicular bodies) (Hicke and Dunn, 2003; Dupre et al, 2004; Bowers and Stevens, 2005). Membrane fusion and vesicular transport are crucial for maintaining a cell's vesicular compartmentalization, which is a defining feature of eukaryotes. It is already known that ubiquitination regulates the initiation of vesicular transport: monoubiquitination leads to the collection of cargo proteins into a vesicle and then to its subsequent release. The vesicle next travels through the cell to its target acceptor membrane, with which it fuses. We present here data which indicate that, in yeast, ubiquitination also regulates the termination of transport—membrane fusion—and does so together with the proteasome, which was not previously known to have a role in fusion. We discovered that fusion of vacuoles cannot proceed unless proteasomal degradation occurs, and that ubiquitinated Ypt7 is a substrate during fusion. Our strongest model suggests that fusion cannot be completed without degradation, by the proteasome, of ubiquitinated Ypt7. To see if the ubiquitin–proteasome system has a role in membrane fusion, we used an in vitro assay for homotypic vacuolar (that is, vacuole-to-vacuole) fusion (Wickner and Haas, 2000; Mayer, 2002; Wickner, 2002). (Lysosomes of Saccharomyces cerevisiae are called ‘vacuoles’). In this model system, AAA-ATPase Sec18/NSF initiates membrane fusion as follows. Sec18 breaks the bonds between the proteins that constitute cis-SNARE complexes, which are located in a membrane, to form free SNARE proteins that are no longer bonded to each other but are still in the membrane. Simultaneously, Sec18/NSF activates Ypt7, a Rab-like GTPase (Haas et al, 1995), via the HOPS chaperone complex; Ypt7 then activates two other GTPases, Rho1 and Cdc42 (Eitzen et al, 2000, 2001; Muller et al, 2001). Docking, an intermediate step in fusion, now occurs: free SNARE proteins on one membrane bond with free SNARE proteins on an opposing membrane, thereby forming trans-SNARE complexes that link opposing membranes together. After vacuoles have docked, Ca2+ release from the vacuolar lumen induces fusion. Many mechanistic details of fusion are still unclear, but it is thought to occur at the vertex ring domain at which the two tightly bound vacuoles meet. Other processes such as vacuolar acidification are also involved in the fusion process, as well as other proteins and lipids such as actin, the Vtc protein complex, vacuolar H+-ATPase, phosphatidylinositol 4,5-biphosphate and ergosterol. Results Indications that the proteasome has a role in vacuolar membrane fusion A multi-copy suppressor screen performed with a proteasomal mutation suggested the hypothesis that the proteasome might be involved in membrane fusion (data not shown). To start evaluating this hypothesis, we studied the in vivo vacuolar morphology of different proteasome mutants to look for evidence of fusion impairment. Using the vital stain FM4-64, we found that one mutant, rpt1-K256S, has an in vivo vacuolar fragmentation phenotype (Figure 1A). Rpt1 is one of six ATPases present in the RP. A point mutation in Rpt1's ATP-binding motif (K256S) causes a slow-growth phenotype with a G1 cell cycle delay (Rubin et al, 1998). Fragmented vacuolar morphology is often observed where membrane fusion is impaired. Thus, the fact that there is a proteasomal mutant with fragmented vacuoles—and so, presumably, impaired fusion—suggests that the proteasome is involved in membrane fusion in vivo. Figure 1.Indications that the proteasome has a role in vacuolar membrane fusion. (A) RPT1 or mutant rpt1-K256S yeast cells were incubated for 1 h with FM4-64 (10 μM), chased for 1.5 h in YPD, and visualized by immunofluorescence microscopy (DY85, DY106). (B) Equal protein amounts of vacuoles and total cell lysate (from wild-type or ecm29Δ cells) were probed with antibodies to visualize the proteasomal Rpn11 and Ecm29 subunits. Vacuoles were washed twice before gel analysis (sMK-172, sMK-173). The bottom panel shows enrichment for the Pho8 marker in both wild-type and ecm29Δ vacuolar preparations (sMK-186, sMK-187). (C) pho8Δ and pep4Δ prb1Δ vacuoles purified from either wild-type or proteasome mutant strains were tested for in vitro fusion activity. We used rpt1-K256S (sMK-191, sMK-193, sMK-220, sMK-230) and pre3-T20A pup1-T30A (sMK-245, sMK-247, sMK-248, sMK-251). Data represent percentage Pho8 activity relative to that from fusion of wild-type vacuoles. Absolute fusion values of RPT1 and PRE3 PUP1 reactions: 0.68 U, 1.67 U. Download figure Download PowerPoint To test if proteasomes are present on vacuoles, we purified vacuoles by equilibrium flotation. (We first verified that equilibrium flotation, our standard procedure for making vacuolar preparations, generates preparations that are highly enriched for vacuoles. We can infer that the procedure does generate such preparations from the behavior of vacuolar protein marker Pho8 (Figure 1B, bottom panel) (Haas, 1995). After washing vacuolar membranes twice, we found them to be rich in proteasomes and in the proteasome-associated Ecm29 protein (Leggett et al, 2002) (Figure 1B; data not shown). The mass of proteasome per unit protein in vacuolar preparations was roughly equivalent to that in total lysates, even though proteasomes are highly abundant in cytosol and nuclear compartments. We incubated vacuoles with the proteasome-specific fluorogenic substrate LLVY-AMC and observed cleavage activity (data not shown), which we would expect to observe if proteasomes were present. As proteasome presence in these preparations depended on the presence of a key vacuolar marker protein (Figure 5), we inferred that proteasomes in the vacuolar preparations are associated with vacuoles, not with nonvacuolar membranes that are contaminants. As it has been proposed that mammalian Ecm29 links proteasomes to membranes (Gorbea et al, 2004), we tested if Ecm29 is a link, in yeast, between proteasomes and vacuoles. We found that it is not: proteasomes were present in equal quantities on wild-type and ecm29Δ vacuoles (Figure 1B). The presence of Ecm29 did not affect vacuolar enrichment (bottom panel, Figure 1B). Figure 2.Inhibition of proteasomal degradation interferes with homotypic vacuolar membrane fusion. (A) Vacuoles from a pho8Δ and a pep4Δ prb1Δ strain (sMK-172, sMK-215) were added to a fusion reaction either alone, or mixed (lanes with black bar). Pho8 activity is depicted in fusion activity units (U). Vacuoles (20 μg) were added to each fusion reaction: either 20 μg of one type of vacuole, or 10 μg each of two types of vacuole. Parallel samples were analyzed by SDS–PAGE for Pho8 maturation. The right part of the panel shows the effects of adding 100 μM proteasome inhibitor PS341, versus carrier DMSO. (B) Three proteasome inhibitors, PS341, MG262 and MG115, were each added to the fusion reaction and compared to the addition of DMSO. In each case, the mixed vacuolar sample's fusion activity is the difference between Pho8 level at 4 and 27°C (sMK-172, sMK-215). Absolute fusion values of reactions without inhibitors: 1.83 U (PS341), 1.66 U (MG262), 1.49 U (MG115). (C) pep4Δ and pho8Δ vacuoles were mixed, lysed in detergent buffer in the presence of either DMSO or 50 μM PS341, incubated for 10 min at 27°C and probed for Pho8 (sMK-395, sMK-404). (D) The proteasome inhibitor Ubistatin-A, which targets multiubiquitin chains on substrates rather than proteasomal active sites, was added to the fusion reaction. Fusion was measured against the DMSO control (sMK-172, sMK-215). Absolute fusion value of reaction without UbistatinA: 1.64 U. Download figure Download PowerPoint We next tested if the rpt1-K256S proteasome mutation, which causes fragmented vacuoles in vivo, interfered with membrane fusion in vitro. We used an assay to compare the degree of fusion that occurs between wild-type vacuoles to that which occurs between mutant vacuoles (Haas, 1995; Wickner and Haas, 2000; Mayer, 2002; Wickner, 2002). This assay causes pep4Δ prb1Δ vacuoles to fuse with pho8Δ vacuoles. Pho8 is initially an inactive pro-enzyme, but it becomes an active alkaline phosphatase when its carboxy-terminus is clipped off by vacuolar proteinases A (Pep4) or B (Prb1). The more fusion activity occurs, the more alkaline phosphatase activity results. The degree of phosphatase activity can then be measured to quantify the degree of membrane fusion (Haas, 1995). This assay showed that there was less fusion activity between the mutant vacuoles (61% less than wild-type vacuoles; Figure 1C), suggesting that the mutant's defect, a defect in the proteasome which leads to fragmented vacuoles in vivo, was physically associated with purified vacuoles in vitro. Although the data suggested a role for the proteasome in fusion, we did not yet know which of its functions is involved. To test if degradation—the proteasome's main function—is involved in fusion, we tested vacuoles purified from a mutant proteasome strain pre3-T20A pup1-T30A (Arendt and Hochstrasser, 1999). This mutant lacks two of the three proteolytic active sites: its trypsin-like and caspase-like sites. Cells containing proteasomes with this mutation have only slight growth impairment, as the proteasomes' major chymotrypsin site is still intact (Arendt and Hochstrasser, 1999). Such cells, with their mild phenotype, did not show in vivo vacuolar fragmentation (data not shown). However, vacuoles purified from these cells exhibited 35% less in vitro vacuolar fusion activity than wild-type vacuoles (Figure 1C). Because less fusion activity occurs when proteasomes are less able to degrade proteins, proteasomal degradation is likely to have a role in fusion. Inhibition of proteasomal degradation interferes with fusion Because the proteasome is essential to life, genetic approaches are limited to studying mutations with partial loss-of-function mutations such as pre3-T20A pup1-T30A. In order to test whether the proteasome's role in fusion includes degradation, it was necessary to interfere more drastically with proteasomes' degradation function, so we switched from genetic to biochemical techniques. The in vitro homotypic vacuolar membrane fusion assay, mentioned earlier, was used to study the effect of proteasome inhibitors on fusion. A typical fusion assay is shown in Figure 2A. Pho8Δ vacuoles were mixed with pep4Δ prb1Δ vacuoles at a fusion-promoting temperature (27°C). This assay measures the level of fusion activity as the difference between the levels of alkaline phosphatase activity that result when pho8Δ and pep4Δ prb1Δ vacuoles are mixed at 27°C and at 4°C (Haas, 1995). The assay worked properly: alkaline phosphatase activity correlated with the amount of clipped mature Pho8 present (Figure 2A), and such activity occurred only when vacuoles of both types were mixed together. Some background alkaline phosphatase activity was observed when pep4Δ prb1Δ vacuoles were incubated alone at 27°C, due to cleavage of pro-enzyme Pho8 from other vacuolar proteases. This background alkaline phosphatase activity did not prevent the assay from measuring fusion because the level of activity was low, correlated with fusion levels and was also sensitive to fusion inhibitors (see below). Figure 3.Ubiquitinated species of Ypt7 and Rho1 are present in vacuolar preparations. (A) Vacuoles from untagged or 3HA-tagged Ypt7 and Rho1 strains in a His6-cMyc-ubiquitin background (SUB592, sMK-303, sMK-307) were purified, subjected to an α-HA immunoprecipitation and probed for both HA and cMyc. (B) His-tagged ubiquitin material from vacuoles (untagged or 3HA-tagged Ypt7 strain: SUB592, sMK-303) was subjected to 2D IEF-SDS–PAGE, and probed for ubiquitin conjugates using an α-cMyc antibody. The top panel (untagged Ypt7) shows three ubiquitin-positive products, labeled A, B1 and B2. The bottom panel uses the 3HA-tagged Ypt7 strain. The products labeled B in the bottom panel are HA-reactive, whereas A is not (data not shown). (C) Vacuoles were purified from a wild-type strain that has unmodified endogenous YPT7- and ubiquitin genes. A fraction of such vacuoles was lysed directly in SDS sample buffer for analysis by SDS–PAGE. This sample lane is labeled ‘vacuolar lysate’. The remaining vacuoles were lysed in NP-40 detergent buffer, and the lysate was used to immunoprecipitate endogenous Ypt7 using the α-Ypt7(g) antibody. Ypt7 was detected by probing with the same α-Ypt7(g) antibody used for immunoprecipitation, as well as with a second, unrelated Ypt7 antibody, α-Ypt7(pep) (sMK-310). (D) Two vacuolar preparations were made from a strain with an unmodified YPT7 gene in a His6-cMyc-ubiquitin background (SUB592). EDTA was either present or absent during the purification from spheroplasting onward. These vacuoles were lysed and subjected to an α-Ypt7(g) immunoprecipitation. These were probed with the α-Ypt7(pep) serum and the cMyc antibody detecting ubiquitin. Download figure Download PowerPoint To test whether proteasome-mediated degradation is involved in vacuolar fusion, we used this assay to measure the effect of proteasome inhibitors on fusion. First, we employed PS341, a selective and potent proteasome inhibitor that belongs to the boronate family and is approved for clinical use. Levels of alkaline phosphatase activity and of mature Pho8 protein were lower when the reaction mixture also contained PS341 (Figure 2A), suggesting that PS341 inhibits vacuolar membrane fusion. Next, we used three distinct lines of experimentation (1–3 below) to confirm that the inhibitor inhibits fusion by targeting the proteasome. (1) To verify that PS341 does not decrease the read-out by targeting the Pep4 and Prb1 proteases, we repeated the experiment described in the previous paragraph with five proteasome inhibitors from a total of three different classes: two boronate inhibitors (PS341, MG262), two aldehyde inhibitors (MG115, MG132) and clasto-lactacystin β-lactone (Figure 2B; data not shown). Inhibitors of different classes all target the proteasome's active sites, which are responsible for degradation, but they use different chemistry to do so. All five were found to interfere with fusion (Figure 2B; data not shown). We used inhibitor concentrations such as 25 or 50 μM that are within the normal range of concentrations used when studying protein substrates. Admittedly, the literature suggests a different range of concentrations, e.g. a range of nM Ki values for boronate inhibitors. However, the ranges suggested in the literature were obtained by the use of peptide substrates: substrates that can be cleaved despite the fact they are not ubiquitinated. In contrast, we worked with protein substrates, which can be degraded only when ubiquitinated. Another difference between such substrates is that it is easier to inhibit degradation of peptides than of proteins, because peptides are usually cleaved by a particular active site of the proteasome, whereas proteins can be cleaved by any of the three (Kisselev et al, 2006). A second and third experiment also suggested that PS341 did not decrease the read-out by targeting the Pep4 and Prb1 proteases. In the second experiment, pep4Δ prb1Δ vacuoles were mixed with pho8Δvacuoles, in the presence of a proteasome inhibitor, by detergent lysis rather than by membrane fusion. In this case, the inhibitor did not prevent Pho8 from maturing (Figure 2C), indicating that the inhibitor did not knockout Pep4 and Prb1. In the third, we pretreated the pep4Δ prb1Δ vacuoles, but not the pho8Δ vacuoles, with the irreversible inhibitor clasto-lactacystin β-lactone. When we mixed these vacuoles in the fusion assay, a low read-out resulted, suggesting that fusion was inhibited (data not shown). Fusion inhibition cannot have been caused by the knockout of the Pep4 and Prb1 proteases, at least in this case, as the pho8Δ vacuoles were not pretreated. (2) If proteasome inhibitors interfere with fusion by inhibiting the proteasome, one would predict that genetic mutations that make the proteasome more susceptible to inhibitors also make membrane fusion more susceptible to inhibitors. To test this prediction, we examined vacuoles from the proteasome mutant pre3-T20A pup1-T30A, which lacks two of its three active sites, for fusion sensitivity to proteasome inhibitors. As predicted, a larger percentage drop in fusion activity was observed when proteasome inhibitors were added to mutant vacuoles than to wild-type vacuoles. For instance, when 25 μM MG132 inhibitor was added to wild-type vacuoles, this reduced fusion activity to 85% of its preinhibited value. By contrast, when the same inhibitor of the same concentration was added to the mutant vacuoles, this reduced fusion activity to 77% of its preinhibited value. Thus, there was an 8% difference between the percentage drops of fusion activity in the wild-type and mutant vacuoles. This 8% difference was observed when the procedure was performed with each of the aldehyde inhibitors MG132 and MG115, using concentrations between 25 and 200 μM. The addition of the boronate inhibitor PS341 resulted in a 5% difference. Similarly, treatment with the irreversible proteasome inhibitor clasto-lactacystin β-lactone resulted in a 17% difference. (3) Experiment (1) used a variety of proteasome inhibitors, each of which works by inhibiting the proteasome's active sites. Experiment (3) instead used a proteasome inhibitor (Ubistatin-A) that works by targeting multiubiquitin chains on substrates, thereby preventing the proteasome from recognizing and degrading them (Verma et al, 2004). We added Ubistatin-A to the in vitro homotypic vacuolar membrane fusion assay. Like the inhibitors used in experiment (1), Ubistatin-A interfered with in vitro vacuolar membrane fusion (Figure 2D). Because the mechanism by which it inhibits is fundamentally different from that of a classic proteasome inhibitor, this is further evidence that the proteasome's function in fusion includes degradation. Discovery and identification of ubiquitin-modified proteins on vacuolar membranes If proteasomal degradation is required for vacuolar fusion, we would expect to find ubiquitin-modified proteins on vacuolar membranes, because virtually all proteasome substrates are ubiquitinated. Using mass spectrometry, we identified such proteins on vacuolar membranes derived from a strain that carries tagged ubiquitin. In more detail, we used a yeast strain in which all four endogenous ubiquitin genes had been replaced with an extrachromosomal plasmid that expresses tagged His6-cMyc-ubiquitin at physiological levels (Finley et al, 1994). This tag allowed us to extract ubiquitinated proteins from purified vacuoles. We analyzed these proteins by mass spectrometry and identified two GTPases, Ypt7 and Rho1, known to have a role in homotypic vacuole fusion (Haas et al, 1995; Eitzen et al, 2000, 2001; Muller et al, 2001). To confirm that Ypt7 and Rho1 exist on vacuoles as multiubiquitinated conjugates, each protein was 3HA-tagged at its amino-terminus and expressed from a GAL promoter in the His6-cMyc-ubiquitin strain. Purified vacuoles from the double-tagged Ypt7 or Rho1 strains were solubilized in detergent and used to perform an α-HA immunoprecipitation (Figure 3A). A substantial portion of the Ypt7 and Rho1 migrated as high molecular weight species, which also tested positive for the cMyc-ubiquitin tag. Figure 4.Identification and description of Two Ypt7 ubiquitination sites. (A) Vacuoles were purified from a 3HA-ypt7 strain (sMK-303). Spectra of two tryptic peptides carrying the -GG modification on lysines K147 and K140 were identified. Lysines K147 and K140 are highlighted in the structure of Ypt7 complexed with GMPP(N)P (Constantinescu et al, 2002). (B) Vacuoles from an untagged (sMK-186), a 3HA-ypt7 tagged (sMK-325) and a 3HA-K147/140R-ypt7 strain (sMK-329) were purified, subjected to an α-HA immunoprecipitation, and probed with an α-Ypt7 antibody. (C) A top view of the Ypt7 structure shown in (A). White circles indicate the two ubiquitination loci on Ypt7. (D) Vacuoles from 3HA-ypt7-tagged strains (K147/140/56/48/5/6R, wild type, K147/140R) were purified, subjected to an α-HA immunoprecipitation and probed with an α-Ypt7 antibody (sMK-395, -325, -329). The bracket indicates ubiquitinated Ypt7 species. (E) This is a side view of the Ypt7 structure presented in (A). The left panel shows Ypt7 complexed with the GTP-analog GMPPNP, the right shows it complexed with GDP. The lysine residues 147 and 140 are highlighted in yellow at the top and 56 near the bottom. The white line shows the distance between the primary amines of lysines 147 and 56 (top panel 35.40 Å, bottom panel 25.46 Å). Download figure Download PowerPoint To determine the number of different ubiquitin-conjugated protein species associated with vacuoles, we analyzed a vacuolar preparation of the His6-cMyc-ubiquitin strain. Ubiquitinated proteins were first eluted from the Ni2+-NTA beads and then separated on a two-dimensional (2D) isoelectric focusing (IEF) (pH 3–10)-SDS–PAGE system. Next, we visualized them by the use of a cMyc-specific antibody. Only a small number of ubiquitin-conjugated species were detected (top panel Figure 3B). However, other ubiquitinated species may exist undetected if they do not focus in the pH 3–10 range or if our protocol did not capture them efficiently. As no proteasome inhibitors were used during vacuole purification, these data suggest that a small number of protein species that are stably multiubiquitinated are associated with vacuolar membranes. To verify that Ypt7 was one of these few ubiquitinated species, we analyzed vacuoles from the 3HA-tagged Ypt7 strain (Figure 3B, bottom panel). A probe of the membrane using an α-HA antibody revealed several ubiquitinated Ypt7 species (products labeled B in Figure 3B, bottom panel) and one species unrelated to Ypt7 (product labeled A in Figure 3B, bottom panel) (data not shown). Product B1 was more abundant in the bottom panel than in the top, consistent with GAL promoter overexpression, and its IEF point was shifted as a result of the fact that B1 became tagged. Product B2 was also more abundant in the bottom than the top panels. Two new species were present in the gel represented by the bottom panel, but not in the gel represented by the top one. Similar results were obtained by the use of an α-ubiquitin antibody and several HA-tagged Ypt7 and Rho1 strains that each expressed wild-type ubiquitin (data not shown). In sum, the 2D analysis showed that Ypt7 is the most prevalent ubiquitinated protein that is present on purified vacuoles. We also identified ubiquitinated Ypt7 species in vacuolar preparations that, like the preparation shown in the top panel of Figure 3B, did not overexpress Ypt7 (Figure 3C). For instance, when we used a preparation from a strain with wild-type ubiquitin and an unmodified YPT7 gene, two α-Ypt7 antibodies independently detected high molecular weight Ypt7 conjugates in vacuolar lysates (Figure 3C). To verify that the conjugates identified by the two different antibodies were both Ypt7, we immunoprecipitated Ypt7 with one antibody and probed the membrane with both. We observed that both antibodies recognized identical 200 kDa Ypt7-positive material, despite the fact that these large conjugates immunoprecipitate inefficiently. The fragility of some ubiquitinated Ypt7 species in some contexts may help to explain why their existence and their presence on membranes have not hitherto been noted. Even though ubiquitinated Ypt7 is stable in the absence of proteasome inhibitors, it can lose its multiubiquitin chain, or split up proteolytically, during the vacuolar purification procedure or subsequent handling. When we performed an experiment using a cMyc-tagged ubiquitin strain that has an unmodified YPT7 gene, we observed that, when EDTA was not present from the spheroplasting stage onwards, some Ypt7 ubiquitinated species were not retained (Figure 3D), although they were retained when EDTA, which has a stabilizing effect, was present. The fact that some, but not all, species of ubiquitinated Ypt7 were EDTA-dependent suggests that they are heterogeneous, probably because their multiubiquitin chains are attached to different sites (see Figure 4). Note that the fraction of Ypt7 that is ubiquitinated was even larger than Figure 3D suggests, because large conjugates precipitate less efficiently than unmodified Ypt7 (Figure 3C). Figure 5.Relationships between Ypt7 species and proteasomes in the context of a vacuolar membrane. (A) Equal protein amounts of purified vacuoles from wild-type and ypt7Δ strains (RG-wt, RG-ypt7Δ) were probed for proteasomes, Ypt7 and Pho8; they were also Coomassie-stained. (B) Vacuoles from wild-type, vps41Δ and vam3Δ strains (RG-wt, RG-vps41Δ, RG-vam3Δ) were probed for Pho8, proteasomes and Ypt7, as in (A). (C) Vacuoles from 3HA-ypt7 tagged strains—wild-type control and K147/140/56/48/5/6R (sMK-413, sMK-421)—were probed for proteasome and Pho8 levels, as in (A). (D) Vacuoles were purified from a wild-type or a proteasomal rpt2-G2A myristoylation site mutant strain. The vacuoles, generated either in the presence or absence of EDTA, were pelleted and either harvested directly, or resuspended and pelleted twice more at 4°C before harvesting. The membranes were analyzed for the presence of both Ypt7 and the proteasome. Indicated are unmodified Ypt7 and high molecular weight Ypt7 ubiquitin conjugates (sMK-309, -310). Download figure Download PowerPoint In sum, we found a small number of ubiquitin-modified proteins on vacuolar membranes. We identified these as the GTPases Ypt7 and Rho1, which are stably multiubiquitinated in most but not all contexts. We decided to focus, in subsequent experiments, on the role of Ypt7 ubiquitination in vacuolar membrane fusion, for several reasons: Ypt7 is necessary for fusion and is the Rab GTPase activ

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThermodynamics of phosphate transfer in the phosphoglucomutase systemErnest J. Peck, Donald S. Kirkpatrick, and William J. RayCite this: Biochemistry 1968, 7, 1, 152–162Publication Date (Print):January 1, 1968Publication History Published online1 May 2002Published inissue 1 January 1968https://doi.org/10.1021/bi00841a020Request reuse permissions Article Views99Altmetric-Citations10LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1008 KB) Get e-Alertsclose Get e-Alerts

<h2>Abstract</h2> The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome–CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation. Expression of the cyclin-dependent kinase 2 (CDK2), itself a downstream target of PI3K/MAPK signaling, leads to increased phosphorylation of RNF157 on the same residues modulated by PI3K and MAPK signaling. Inhibition of PI3K and MEK in combination or of CDK2 by their respective small-molecule inhibitors reduces RNF157 phosphorylation at these residues and attenuates RNF157 interaction with CDH1 and its subsequent degradation. Knockdown of endogenous RNF157 in melanoma cells leads to late S phase and G₂/M arrest and induces apoptosis, the latter further potentiated by concurrent PI3K/MEK inhibition, consistent with a role for RNF157 in the cell cycle. We propose that RNF157 serves as a novel node integrating oncogenic signaling pathways with the cell cycle machinery and promoting optimal cell cycle progression in transformed cells.

Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn’s disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri) , a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo . Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.

Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.

Triggered by Offset, Multiplexed, Accurate mass, High resolution, and Absolute Quantitation (TOMAHAQ) is a recently introduced targeted proteomics method that combines peptide and sample multiplexing. TOMAHAQ assays enable sensitive and accurate multiplexed quantification by implementing an intricate data collection scheme that comprises multiple MSn scans, mass inclusion lists, and data-driven filters. Consequently, manual creation of TOMAHAQ methods can be time-consuming and error prone, while the resulting TOMAHAQ data may not be compatible with common mass spectrometry analysis pipelines. To address these concerns we introduce TomahaqCompanion, an open-source desktop application that enables rapid creation of TOMAHAQ methods and analysis of TOMAHAQ data. Starting from a list of peptide sequences, a user can perform each step of TOMAHAQ assay development including (1) generation of priming run target list, (2) analysis of priming run data, (3) generation of TOMAHAQ method file, and (4) analysis and export of quantitative TOMAHAQ data. We demonstrate the flexibility of TomahaqCompanion by creating a variety of methods testing TOMAHAQ parameters (e.g., number of SPS notches, run length, etc.). Lastly, we analyze an interference sample comprising heavy yeast peptides, a standard human peptide mixture, TMT11-plex, and super heavy TMT (shTMT) isobaric labels to demonstrate ∼10–200 attomol limit of quantification within a complex background using TOMAHAQ.

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor-related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose-response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCR α ) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCR α occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCR α in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCR α is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCR α peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCR α remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.

Abstract The ability to quantitatively measure a small molecule’s interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.

Summary We observed congruent contemporaneous concordance of malignant melanoma in identical brothers of a set of triplets. Four and one-half years after surgery, one twin had metastatic disease and the other was apparently free of tumor. Blastoid transformation of lymphocytes measured by DNA synthesis and cytotoxicity of lymphocytes for melanoma cells measured by fluorescein diacetate loss in mixed lymphocyte-tumor cell cultures were both substantially greater with lymphocytes from the tumor-free twin. Since the brothers were monozygous, this cannot be attributed to transplantation-antigenic differences. Possible explanations include neoantigenization of the tumor and/or depletion or blocking of sensitized lymphocytes from the autochthonous host. The data extend the evidence for immunity against tumors in man and the techniques for demonstrating it.

In these experiments precision-cut tissue slices from two existing transgenic mouse strains, with transgenes that couple promoting or binding elements to a reporter protein, were used for determination of reporter induction. This approach combines the power of transgenic animals with the practicality of in vitro systems to investigate the biological impact of xenobiotics. Additionally, the normal cellular architecture and heterogeneity is retained in precision-cut tissue slices. Two transgenic mouse strains, one of which couples the promoting region of CYP 1A1 to beta-galactosidase, and another which couples two forward and two backward 12-O-tetradecanoyl phorbol-13-acetate (TPA) repeat elements (TRE) to luciferase (termed AP-1/luciferase), were used to determine the feasibility of this approach. Precision-cut kidney and liver slices from both transgenic strains remain viable as determined by slice K(+) ion content and LDH enzyme release. Liver slices harvested from the CYP 1A1/beta-galactosidase transgenic mice exhibit a 14-fold increase in beta-galactosidase activity when incubated with beta-napthoflavone for 24 h. Kidney and liver slices obtained from the AP-1/luciferase transgenic mice demonstrate induction of luciferase (up to 2.5-fold) when incubated with phorbol myristate acetate (PMA or TPA) up to 4 h. These data indicate that precision-cut tissue slices from transgenic mice offer a novel in vitro method for toxicity evaluation while maintaining normal cell heterogeneity.

The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899).

1. After separation by SDS gel-chromatography, analysis of AEP-containing glycoproteins from M. senile, indicated 66% amino acids with 220 AEP res./1000 res. and 30% carbohydrate for high mol. wt (greater than 10(7) forms and 80% amino acids with 25-50 AEP res./1000 res. and 10% carbohydrate for low mol. wt (2-4 x 10(4) forms. 2. Uronic acids, sulfate, lipid, and sialic acids were absent. 3. Mild base digestion released AEP-hexosamine containing oligosaccharides and destroyed ser-thr residues in the high mol. wt components. 4. Phosphonoglycoproteins appear to be acidic connective tissue components with AEP linked to hexosamine containing oligosaccharide side chains.

Abstract 1. 1. The amino acid compositions of the skin collagens from Rana catesbiana larva and adult and from the skin and tail of Notophthalmus viridescens were determined. 2. 2. The melting temperatures of the collagens were found to be 27°C for the tadpole; 29·3° for the frog and 29·8° for the newt. 3. 3. Imino acid content correlated with the T m of newt collagen. The T m s of the anuran collagens did not correlate with imino acid content. 4. 4. Using two methods of analysis, no significant tryptophan was detected in the anuran collagens. Tryptophan equivalent to one residue per tropocollagen molecule was detectable in the urodele collagens.

ABSTRACT Peg10 (paternally expressed gene 10) is an imprinted gene that is essential for placental development. It is thought to derive from a Ty3-gyspy LTR (long terminal repeat) retrotransposon and retains Gag and Pol-like domains. Here we show that the Gag domain of PEG10 can promote vesicle budding similar to the HIV p24 Gag protein. Expressed in a subset of mouse endocrine organs in addition to the placenta, PEG10 was identified as a substrate of the deubiquitinating enzyme USP9X. Consistent with PEG10 having a critical role in placental development, PEG10-deficient trophoblast stem cells (TSCs) exhibited impaired differentiation into placental lineages. PEG10 expressed in wild-type, differentiating TSCs was bound to many cellular RNAs including Hbegf (Heparin-binding EGF-like growth factor), which is known to play an important role in placentation. Expression of Hbegf was reduced in PEG10-deficient TSCs suggesting that PEG10 might bind to and stabilize RNAs that are critical for normal placental development.

&lt;div&gt;Abstract&lt;p&gt;&lt;i&gt;PIK3CA&lt;/i&gt; is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with &lt;i&gt;PIK3CA&lt;/i&gt;-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer.&lt;/p&gt;Significance:&lt;p&gt;The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in &lt;i&gt;PIK3CA&lt;/i&gt;-mutant tumors.&lt;/p&gt;&lt;p&gt;&lt;i&gt;&lt;a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-21-1411" target="_blank"&gt;See related commentary by Vanhaesebroeck et al., p. 20&lt;/a&gt;.&lt;/i&gt;&lt;/p&gt;&lt;p&gt;&lt;i&gt;&lt;a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-12-1-ITI" target="_blank"&gt;This article is highlighted in the In This Issue feature, p. 1&lt;/a&gt;&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;