Heiko M. Möller

The inhibition of carbohydrate-protein interactions by tailored multivalent ligands is a powerful strategy for the treatment of many human diseases. Crucial for the success of this approach is an understanding of the molecular mechanisms as to how a binding enhancement of a multivalent ligand is achieved. We have synthesized a series of multivalent N-acetylglucosamine (GlcNAc) derivatives and studied their interaction with the plant lectin wheat germ agglutinin (WGA) by an enzyme-linked lectin assay (ELLA) and X-ray crystallography. The solution conformation of one ligand was determined by NMR spectroscopy. Employing a GlcNAc carbamate motif with alpha-configuration and by systematic variation of the spacer length, we were able to identify divalent ligands with unprecedented high WGA binding potency. The best divalent ligand has an IC(50) value of 9.8 microM (ELLA) corresponding to a relative potency of 2350 (1170 on a valency-corrected basis, i.e., per mol sugar contained) compared to free GlcNAc. X-ray crystallography of the complex of WGA and the second best, closely related divalent ligand explains this activity. Four divalent molecules simultaneously bind to WGA with each ligand bridging adjacent binding sites. This shows for the first time that all eight sugar binding sites of the WGA dimer are simultaneously functional. We also report a tetravalent neoglycopeptide with an IC(50) value of 0.9 microM being 25,500 times higher than that of GlcNAc (6400 times per contained sugar) and the X-ray structure analysis of its complex with glutaraldehyde-cross-linked WGA. Comparison of the crystal structure and the solution NMR structure of the neoglycopeptide as well as results from the ELLA suggest that the conformation of the glycopeptide in solution is already preorganized in a way supporting multivalent binding to the protein. Our findings show that bridging adjacent protein binding sites by multivalent ligands is a valid strategy to find high-affinity protein ligands and that even subtle changes of the linker structure can have a significant impact on the binding affinity.

By a stepwise synthesis strategy biofunctionalized Pyrrolopyrrole Cyanines (PPCy) with an asymmetric substitution pattern were obtained. These exhibit extremely strong and narrowband NIR absorption and fluorescence. Internalization of a peptide bound PPCy is demonstrated using live cell microscopy.

The treatment of infections due to the opportunistic pathogen Pseudomonas aeruginosa is often difficult, as a consequence of bacterial biofilm formation. Such a protective environment shields the bacterium from host defense and antibiotic treatment and secures its survival. One crucial factor for maintenance of the biofilm architecture is the carbohydrate-binding lectin LecB. Here, we report the identification of potent mannose-based LecB inhibitors from a screening of four series of mannosides in a novel competitive binding assay for LecB. Cinnamide and sulfonamide derivatives are inhibitors of bacterial adhesion with up to a 20-fold increase in affinity to LecB compared to the natural ligand methyl mannoside. Because many lectins of the host require terminal saccharides (e.g., fucosides), such capped structures as reported here may offer a beneficial selectivity profile for the pathogenic lectin. Both classes of compounds show distinct binding modes at the protein, offering the advantage of a simultaneous development of two new lead structures as anti-pseudomonadal drugs with an anti-virulence mode of action.

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

The process for selecting potent and effective carbohydrate antigens is not well-established. A combination of synthetic glycan microarray screening, surface plasmon resonance analysis, and saturation transfer difference NMR spectroscopy was used to dissect the antibody-binding surface of a carbohydrate antigen, revealing crucial binding elements with atomic-level detail. This analysis takes the first step toward uncovering the rules for structure-based design of carbohydrate antigens.

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC-1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O-alpha-D-GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build-up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N-terminal PDT segment whereas the C-terminal RP segment has fewer interactions with the protein. In contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N-acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N-terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.

Hepatitis C virus (HCV) core protein forms the internal viral coat that encapsidates the genomic RNA and is enveloped in a host cell-derived lipid membrane. As the single capsid protein, core should be capable of multimerization but attempts to produce virus-like particles following expression of HCV structural proteins have not been successful. In this study, we have analysed the interaction capacity of full-length and truncated HCV core using the yeast two-hybrid system. Full-length core containing or lacking the translocation signal for the E1 glycoprotein did not interact with full-length or truncated core proteins. Truncation to the N-terminal 122 aa revealed an interaction domain which was mapped to the tryptophan-rich sequence from aa 82-102 and was termed the main homotypic interaction domain. The C-terminal hydrophobic fragment of core (aa 122-172) was incapable of interacting with itself but interacted with the main homotypic interaction domain in trans (the weak heterotypic interaction domain). Core proteins truncated at their N and C termini (aa 46-102) were trans-activating when fused to the DNA-binding domain of GAL4. Based on our results, we suggest that the C-terminal segment may interact in cis with the main homotypic interaction domain and thereby prevent multimerization. Core-core interaction was also observed for in vitro-translated proteins bound to truncated immobilized core 102. However, interaction was less specific in this system suggesting that protein interaction and possibly conformational alteration of core may be dependent on the experimental system.

Bacterial degradation of steroids is widespread, but the metabolic pathways have rarely been explored. Previous studies with Pseudomonas sp. strain Chol1 and the C(24) steroid cholate have shown that cholate degradation proceeds via oxidation of the A ring, followed by cleavage of the C(5) acyl side chain attached to C-17, with 7α,12β-dihydroxy-androsta-1,4-diene-3,17-dione (12β-DHADD) as the product. In this study, the pathway for degradation of the acyl side chain of cholate was investigated in vitro with cell extracts of strain Chol1. For this, intermediates of cholate degradation were produced with mutants of strain Chol1 and submitted to enzymatic assays containing coenzyme A (CoA), ATP, and NAD(+) as cosubstrates. When the C(24) steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO) was used as the substrate, it was completely transformed to 12α-DHADD and 7α-hydroxy-androsta-1,4-diene-3,12,17-trione (HADT) as end products, indicating complete removal of the acyl side chain. The same products were formed with the C(22) steroid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) as the substrate. The 12-keto compound HADT was transformed into 12β-DHADD in an NADPH-dependent reaction. When NAD(+) was omitted from assays with DHOCTO, a new product, identified as 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA), was formed. This aldehyde was transformed to DHOPDC and DHOPDC-CoA in the presence of NAD(+), CoA, and ATP. These results revealed that degradation of the C(5) acyl side chain of cholate does not proceed via classical β-oxidation but via a free aldehyde that is oxidized to the corresponding acid. The reaction leading to the aldehyde is presumably catalyzed by an aldolase encoded by the gene skt, which was previously predicted to be a β-ketothiolase.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen.

1-Methyl-4-phenyl-tetrahydropyridine (MPTP) is among the most widely used neurotoxins for inducing experimental parkinsonism. MPTP causes parkinsonian symptoms in mice, primates, and humans by killing a subpopulation of dopaminergic neurons. Extrapolations of data obtained using MPTP-based parkinsonism models to human disease are common; however, the precise mechanism by which MPTP is converted into its active neurotoxic metabolite, 1-methyl-4-phenyl-pyridinium (MPP(+)), has not been fully elucidated. In this study, we aimed to address two unanswered questions related to MPTP toxicology: (1) Why are MPTP-converting astrocytes largely spared from toxicity? (2) How does MPP(+) reach the extracellular space?In MPTP-treated astrocytes, we discovered that the membrane-impermeable MPP(+), which is generally assumed to be formed inside astrocytes, is almost exclusively detected outside of these cells. Instead of a transporter-mediated export, we found that the intermediate, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)), and/or its uncharged conjugate base passively diffused across cell membranes and that MPP(+) was formed predominately by the extracellular oxidation of MPDP(+) into MPP(+). This nonenzymatic extracellular conversion of MPDP(+) was promoted by O2, a more alkaline pH, and dopamine autoxidation products.Our data indicate that MPTP metabolism is compartmentalized between intracellular and extracellular environments, explain the absence of toxicity in MPTP-converting astrocytes, and provide a rationale for the preferential formation of MPP(+) in the extracellular space. The mechanism of transporter-independent extracellular MPP(+) formation described here indicates that extracellular genesis of MPP(+) from MPDP is a necessary prerequisite for the selective uptake of this toxin by catecholaminergic neurons.

The bile salts cholate, deoxycholate, chenodeoxycholate and lithocholate are released from vertebrates into soil and water where environmental bacteria degrade these widespread steroid compounds. It was investigated whether different enzymes are required for the degradation of these tri-, di- and monohydroxylated bile salts in the model organism Pseudomonas sp. strain Chol1. Experiments with available and novel mutants showed that the degradation of the C5 -carboxylic side chain attached to the steroid skeleton is catalysed by the same set of enzymes. A difference was found for the degradation of partially degraded bile salts consisting of H-methylhexahydroindanone-propanoates (HIPs). With deoxycholate and lithocholate, which lack a hydroxy group at C7 of the steroid skeleton, an additional acyl-coenzyme A (CoA) dehydrogenase was required for β-oxidation of the C3 -carboxylic side chain attached to the methylhexahydroindanone moiety. The β-oxidation of this side chain could be measured in vitro. With cholate and deoxycholate, a reductive dehydroxylation of the C12-hydroxy group of HIP was required. Deletion of candidate genes for this reaction step revealed that a so-far unknown steroid dehydratase and a steroid oxidoreductase were responsible for this CoA-dependent reaction. These results showed that all bile salts are channelled into a common pathway via bypass reactions with 3'-hydroxy-HIP-CoA as central intermediate.

Abstract Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.

Abstract Background The SPFH protein superfamily is a diverse family of proteins whose eukaryotic members are involved in the scaffolding of detergent-resistant microdomains. Recently the origin of the SPFH proteins has been questioned. Instead, convergent evolution has been proposed. However, an independent, convergent evolution of three large prokaryotic and three eukaryotic families is highly unlikely, especially when other mechanisms such as lateral gene transfer which could also explain their distribution pattern have not yet been considered. To gain better insight into this very diverse protein family, we have analyzed the genomes of 497 microorganisms and investigated the pattern of occurrence as well as the genomic vicinity of the prokaryotic SPFH members. Results According to sequence and operon structure, a clear division into 12 subfamilies was evident. Three subfamilies (SPFH1, SPFH2 and SPFH5) show a conserved operon structure and two additional subfamilies are linked to those three through functional aspects (SPFH1, SPFH3, SPFH4: interaction with FtsH protease). Therefore these subgroups most likely share common ancestry. The complex pattern of occurrence among the different phyla is indicative of lateral gene transfer. Organisms that do not possess a single SPFH protein are almost exclusively endosymbionts or endoparasites. Conclusion The conserved operon structure and functional similarities suggest that at least 5 subfamilies that encompass almost 75% of all prokaryotic SPFH members share a common origin. Their similarity to the different eukaryotic SPFH families, as well as functional similarities, suggests that the eukaryotic SPFH families originated from different prokaryotic SPFH families rather than one. This explains the difficulties in obtaining a consistent phylogenetic tree of the eukaryotic SPFH members. Phylogenetic evidence points towards lateral gene transfer as one source of the very diverse patterns of occurrence in bacterial species.

Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA. Na+ is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR) as the first complex in its respiratory chain. The Na+-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na+ translocation by the Na+-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na+-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na+-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.

Summary The distribution and the metabolic pathways of bacteria degrading steroid compounds released by eukaryotic organisms were investigated using the bile salt cholate as model substrate. Cholate‐degrading bacteria could be readily isolated from freshwater environments. All isolated strains transiently released steroid degradation intermediates into culture supernatants before their further degradation. Cholate degradation could be initiated via two different reaction sequences. Most strains degraded cholate via a reaction sequence known from the model organism P seudomonas sp. strain Chol 1 releasing intermediates with a 3‐keto‐Δ 1,4 ‐diene structure of the steroid skeleton. The actinobacterium D ietzia sp. strain Chol 2 degraded cholate via a different and yet unexplored reaction sequence releasing intermediates with a 3‐keto‐Δ 4,6 ‐diene‐7‐deoxy structure of the steroid skeleton such as 3,12‐dioxo‐4,6‐choldienoic acid ( DOCDA ). Using DOCDA as substrate, two Alphaproteobacteria , strains Chol 10–11, were isolated that produced the same cholate degradation intermediates as strain Chol 2. With DOCDA as substrate for P seudomonas sp. strain Chol 1 only the side chain was degraded while the ring system was transformed into novel steroid compounds accumulating as dead‐end metabolites. These metabolites could be degraded by the DOCDA ‐producing strains Chol 10–11. These results indicate that bacteria with potentially different pathways for cholate degradation coexist in natural habitats and may interact via interspecies cross‐feeding.

We present a divergent strategy for the fluorination of phenylacetic acid derivatives that is induced by a charge-transfer complex between Selectfluor and 4-(dimethylamino)pyridine. A comprehensive investigation of the conditions revealed a critical role of the solvent on the reaction outcome. In the presence of water, decarboxylative fluorination through a single-electron oxidation is dominant. Non-aqueous conditions result in the clean formation of α-fluoro-α-arylcarboxylic acids.

A series of six mono-, di-, and trivalent N,N'-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II)-catalyzed diazo transfer and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA) from Triticum vulgaris by an enzyme-linked lectin assay (ELLA) employing covalently immobilized N-acetylglucosamine (GlcNAc) as a reference ligand. IC(50) values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N'-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC(50) value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR.Background: The Na+-NQR is a respiratory Na+ pump found in prokaryotes.Results: The NqrA subunit binds two quinone-type ligands in direct vicinity to each other.Conclusion: Simultaneous binding of two quinones enhances catalytic efficiency of the final electron transfer step.Significance: We provide the first direct experimental evidence of simultaneous quinone binding with relevance for the catalytic mechanism. The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR. Background: The Na+-NQR is a respiratory Na+ pump found in prokaryotes. Results: The NqrA subunit binds two quinone-type ligands in direct vicinity to each other. Conclusion: Simultaneous binding of two quinones enhances catalytic efficiency of the final electron transfer step. Significance: We provide the first direct experimental evidence of simultaneous quinone binding with relevance for the catalytic mechanism.

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa. Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa. Ecdysteroids are a large family of structurally diverse polyhydroxylated sterols found in plants and animals of the Arthropoda phylum (reviewed in Ref. 1Gilbert L.I. Insect Endocrinology. 1st edition. Elsevier/Academic Press, London; Waltham, MA2012Google Scholar). In insects, ecdysteroids control life-cycle progression, including molting, reproduction, stress response, and lifespan, via conserved regulatory pathways (reviewed in Ref. 2Schwedes C.C. Carney G.E. Ecdysone signaling in adult Drosophila melanogaster.J. Insect Physiol. 2012; 58: 293-302Crossref PubMed Scopus (108) Google Scholar). The fruit fly Drosophila melanogaster is an established model organism in developmental biology and now is becoming an important system for studying lipid metabolism and its hormonal regulation (3Baker K.D. Thummel C.S. Diabetic larvae and obese flies-emerging studies of metabolism in Drosophila.Cell Metab. 2007; 6: 257-266Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 4Brogiolo W. Stocker H. Ikeya T. Rintelen F. Fernandez R. Hafen E. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control.Curr. Biol. 2001; 11: 213-221Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar, 5Spindler K.D. Honl C. Tremmel C. Braun S. Ruff H. Spindler-Barth M. Ecdysteroid hormone action.Cell. Mol. Life Sci. 2009; 66: 3837-3850Crossref PubMed Scopus (91) Google Scholar–6Gutierrez E. Wiggins D. Fielding B. Gould A.P. Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.Nature. 2007; 445: 275-280Crossref PubMed Scopus (297) Google Scholar). Drosophila is a sterol auxotroph, and its steroidogenesis relies entirely upon dietary sources of sterols. However, the exact molecular mechanisms by which the availability of dietary sterols alters the ecdysteroids profile and affects fly development remain poorly understood (7Carvalho M. Schwudke D. Sampaio J.L. Palm W. Riezman I. Dey G. Gupta G.D. Mayor S. Riezman H. Shevchenko A. et al.Survival strategies of a sterol auxotroph.Development. 2010; 137: 3675-3685Crossref PubMed Scopus (105) Google Scholar). Ecdysone (E), 20-hydroxyecdysone (20H), and makisterone A (Fig. 1 and supplementary Fig. I) are major ecdysteroids in Drosophila. Their content in individual animals depends on both the developmental stage and the availability of dietary sterols (8Blais C. Blasco T. Maria A. Dauphin-Villemant C. Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010; 878: 925-932Crossref PubMed Scopus (20) Google Scholar, 9Redfern C.P. Evidence for the presence of makisterone A in Drosophila larvae and the secretion of 20-deoxymakisterone A by the ring gland.Proc. Natl. Acad. Sci. USA. 1984; 81: 5643-5647Crossref PubMed Google Scholar). Apart from these three major hormones, several low abundant molecules, including 20-deoxy-makisterone A (9Redfern C.P. Evidence for the presence of makisterone A in Drosophila larvae and the secretion of 20-deoxymakisterone A by the ring gland.Proc. Natl. Acad. Sci. USA. 1984; 81: 5643-5647Crossref PubMed Google Scholar), 24-epi-20-deoxy-makisterone A (8Blais C. Blasco T. Maria A. Dauphin-Villemant C. Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010; 878: 925-932Crossref PubMed Scopus (20) Google Scholar), and 20-hydroxyecdysone-22-acetate (10Maroy P. Kaufmann G. Dubendorfer A. Embryonic ecdysteroids of Drosophila melanogaster.J. Insect Physiol. 1988; 34: 633-637Crossref Scopus (55) Google Scholar) have been characterized; the existence of other ecdysteroid-related structures has been inferred from indirect biological evidence (11Guittard E. Blais C. Maria A. Parvy J.P. Pasricha S. Lumb C. Lafont R. Daborn P.J. Dauphin-Villemant C. CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.Dev. Biol. 2011; 349: 35-45Crossref PubMed Scopus (133) Google Scholar). Furthermore, previous observations indicated that ecdysteroids may be present in a variety of conjugates with phosphate, sulfate, fatty acid, and carbohydrate moieties serving as storage or transport forms of active hormones (12Dubendorfer A. Maroy P. Ecdysteroid conjugation by tissues of adult females of Drosophila melanogaster.Insect Biochem. 1986; 16: 109-113Crossref Scopus (21) Google Scholar, 13Wigglesworth K.P. Lewis D. Rees H.H. Ecdysteroid titer and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae).Arch. Insect Biochem. Physiol. 1985; 2: 39-54Crossref Scopus (56) Google Scholar, 14Sonobe H. Ito Y. Phosphoconjugation and dephosphorylation reactions of steroid hormone in insects.Mol. Cell. Endocrinol. 2009; 307: 25-35Crossref PubMed Scopus (15) Google Scholar, 15Isaac R.E. Desmond H.P. Rees H.H. Isolation and identification of 3-acetylecdysone 2-phosphate, a metabolite of ecdysone, from developing eggs of Schistocera gregaria.Biochem. J. 1984; 217: 239-243Crossref PubMed Scopus (14) Google Scholar–16Thompson M.J. Feldlaufer M.F. Lozano R. Rees H.H. Lusby W.R. Svoboda J.A. Wilzer K.R. Metabolism of 26-[C-14]hydroxyecdysone 26-phosphate in the tobacco hornworm, Manduca sexta L., to a new ecdysteroid conjugate - 26-[C-14]hydroxyecdysone 22-glucoside.Arch. Insect Biochem. Physiol. 1987; 4: 1-15Crossref Scopus (26) Google Scholar). Depending on the stage of development, a single Drosophila larva might contain from 50 to 400 pg/mg of ecdysteroids in total (17Kozlova T. Thummel C.S. Steroid regulation of postembryonic development and reproduction in Drosophila.Trends Endocrinol. Metab. 2000; 11: 276-280Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 18Parvy J.P. Blais C. Bernard F. Warren J.T. Petryk A. Gilbert L.I. O'Connor M.B. Dauphin-Villemant C. A role for betaFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.Dev. Biol. 2005; 282: 84-94Crossref PubMed Scopus (107) Google Scholar), along with over one billion-fold molar excess of structural (glycerophospholipid, ceramide, and sterol) and energy storage (di- and triacylglycerol) lipids. Radioimmunoassay (RIA) has historically been most common way of quantifying ecdysteroids in insects having small body size (e.g., Drosophila). However, RIA offers limited dynamic range and, not being able to distinguish individual ecdysteroids, may only report the total content of all ecdysone-related molecules. Methods have been developed for detecting individual endogenous insect ecdysteroids by GC-MS (19Le Bizec B. Antignac J-P. Monteau F. Andre F. Ecdysteroids: one potential new anabolic family in breeding animals.Anal. Chim. Acta. 2002; 473: 89-97Crossref Scopus (32) Google Scholar) and LC-MS/MS (8Blais C. Blasco T. Maria A. Dauphin-Villemant C. Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010; 878: 925-932Crossref PubMed Scopus (20) Google Scholar, 20Li Y. Warren J.T. Boysen G. Gilbert L.I. Gold A. Sangaiah R. Ball L.M. Swenberg J.A. Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry.Rapid Commun. Mass Spectrom. 2006; 20: 185-192Crossref PubMed Scopus (17) Google Scholar); however, they appeared to be far less sensitive compared with RIA. Ecdysteroids are detected by electrospray ionization mass spectrometry as protonated molecular cations (8Blais C. Blasco T. Maria A. Dauphin-Villemant C. Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010; 878: 925-932Crossref PubMed Scopus (20) Google Scholar, 20Li Y. Warren J.T. Boysen G. Gilbert L.I. Gold A. Sangaiah R. Ball L.M. Swenberg J.A. Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry.Rapid Commun. Mass Spectrom. 2006; 20: 185-192Crossref PubMed Scopus (17) Google Scholar). However, their ionization capacity is low compared with major glycerophospholids, such as phosphatidylcholines and phosphatidylethanolamines. MS/MS spectra of ecdysteroids are dominated by abundant peaks of water-loss fragments (21Wang Y.H. Avula B. Jadhav A.N. Smillie T.J. Khan I.A. Structural characterization and identification of ecdysteroids from Sida rhombifolia L. in positive electrospray ionization by tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2008; 22: 2413-2422Crossref PubMed Scopus (19) Google Scholar, 22Stevens J.F. Reed R.L. Morre J.T. Characterization of phytoecdysteroid glycosides in Meadowfoam (Limnanthes alba) seed meal by positive and negative ion LC-MS/MS.J. Agric. Food Chem. 2008; 56: 3945-3952Crossref PubMed Scopus (30) Google Scholar), which is common for small relatively hydrophilic molecules. Therefore, unequivocal identification of known and discovery of novel endogenous ecdysteroids require alternative approaches relying on molecular forms having more specific fragmentation pathways. Despite their structural heterogeneity, ecdysteroids share a few common features that distinguish them from the majority of oxysterols (Fig. 1). First, they are polyhydroxylated: an ecdysteroid may comprise up to six hydroxyl groups. Second, they possess the 6-ketone group conjugated with the C7-C8 double bond along with the vinyl hydroxyl at C14. Therefore, it is conceivable that derivatizing the 6-ketone group with an appropriate reagent might enhance the ionization capacity and increase the analysis specificity of the entire ecdysteroid complement. Girard reagents (23Girard A. Sandulesco G. On a new series of reactants of the carbonyl group, their use for the extraction of ketonic substances and for the microchemical characterisation of aldehydes and ketones.Helv. Chim. Acta. 1936; 19: 1096-1107Google Scholar), a family of quaternary ammonium hydrazides (24Wheeler O.H. The Girard reagents.J. Chem. Educ. 1968; 45: 435-437Crossref Scopus (54) Google Scholar) typically comprising pyridine or trimethylamine (TMA) moieties, were employed for the derivatization of 3-ketone groups in mammalian oxysterols produced by their enzymatic oxidation (25Karu K. Hornshaw M. Woffendin G. Bodin K. Hamberg M. Alvelius G. Sjovall J. Turton J. Wang Y. Griffiths W.J. Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols.J. Lipid Res. 2007; 48: 976-987Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Wang Y. Sousa K.M. Bodin K. Theofilopoulos S. Sacchetti P. Hornshaw M. Woffendin G. Karu K. Sjovall J. Arenas E. et al.Targeted lipidomic analysis of oxysterols in the embryonic central nervous system.Mol. Biosyst. 2009; 5: 529-541Crossref PubMed Scopus (31) Google Scholar, 27Griffiths W.J. Wang Y. Alvelius G. Liu S. Bodin K. Sjovall J. Analysis of oxysterols by electrospray tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 2006; 17: 341-362Crossref PubMed Scopus (95) Google Scholar–28Shackleton C.H. Chuang H. Kim J. de la Torre X. Segura J. Electrospray mass spectrometry of testosterone esters: potential for use in doping control.Steroids. 1997; 62: 523-529Crossref PubMed Scopus (132) Google Scholar): converting charge-neutral oxysterols to strongly positively charged hydrazones enhanced their solubility and ionization capacity (an approach recently termed “charge-tagging”) (29Meljon A. Theofilopoulos S. Shackleton C.H. Watson G.L. Javitt N.B. Knolker H.J. Saini R. Arenas E. Wang Y. Griffiths W.J. Analysis of bioactive oxysterols in newborn mouse brain by LC/MS.J. Lipid Res. 2012; 53: 2469-2483Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). If subjected to MS/MS, Girard hydrazones undergo facile neutral loss of pyridine or TMA. Net positive charge is then transferred to the sterol backbone, and upon collision-induced dissociation (CID), several structure-specific informative fragments are produced. These oxidized oxysterols, however, are different from ecdysteroids: their ketone group is located at the outer A-ring, which might ease constraints imposed by sterical hindrance (24Wheeler O.H. The Girard reagents.J. Chem. Educ. 1968; 45: 435-437Crossref Scopus (54) Google Scholar); additionally, there is no vinyl hydroxyl group next to the double bond within the B-ring. Oxysterols have fewer hydroxyl moieties and are generally less prone to water losses. Here we demonstrate that a combination of Girard derivatization and LC-MS/MS enabled simultaneous detection of major ecdysteroids at the endogenous (picogram) level in the extract of a single Drosophila pupa. It paves the way toward the rapid comprehensive characterization of the ecdysteroidome - a full complement of ecdysteroids, their precursors, intermediate conjugates, and metabolites present in a developing fly. Standards of 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone, and polypodine B were purchased from Sigma-Aldrich Chemie (Munich, Germany); ecdysone from A.G. Scientific (Göttingen, Germany); muristerone A from Merck Biosciences (Bad Soden, Germany); and makisterone A and ponasterone A from Enzo Life Sciences (Lörrach, Germany). All solvents were LC-MS grade. Girard T [1-(carboxymethyl)trimethylammonium chloride hydrazide] and Girard C (potassium hydrazincarbonyl acetate) were purchased from Sigma-Aldrich Chemie; and Girard P [1-(carboxymethyl)pyridinium chloride hydrazide] from TCI Europe (Eschborn, Germany). HPLC was performed on Agilent 1200 system equipped with a trap column (OPTI-PAK, 1 µl, C18) from Dichrom GmbH (Marl, Germany) that was mounted in-line to a 0.5 mm × 150 mm analytical column packed with Zorbax SB-C18 5 μm (Böblingen, Germany). The mobile phase consisted of solvent A (0.1% aqueous formic acid) and solvent B (0.1% formic acid in neat acetonitrile). Gradient elution program was as follows: delivering 5% of B during first 10 min until the sample is loaded and concentrated on the trap column; ramping from 15% to 30% of B between 11 min to 30 min; increasing up to 100% of B in 1 min and holding for 9 min; stepping down to 5% of B in 1 min and holding for 19 min to equilibrate the column to starting conditions. The flow rate was 10 μl/min; injection volumes are specified for each experiment. For direct infusion experiments, the reaction mixture was cleaned up by solid phase extraction on Oasis HLB (6 cc/150 mg) cartridges from Waters (Eschborn, Germany), dried down in a vacuum centrifuge, and redissolved in isopropanol/methanol/chloroform 4:2:1 (v/v/v) containing 7.5 mM ammonium acetate. MS and MS/MS spectra were acquired on a Q Exactive tandem mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nanoflow ion source TriVersa NanoMate (Advion BioSciences, Ithaca, NY). Gas backpressure was 1.25 psi, and ionization voltage was 0.95 kV. Spectra were acquired in MS mode with the target mass resolution of Rm/z 200= 140,000 under automated gain control (AGC) target value of 1,000,000 and maximum injection time of 1 s. Fragmentation was performed in the HCD cell (30Schuhmann K. Herzog R. Schwudke D. Metelmann-Strupat W. Bornstein S.R. Shevchenko A. Bottom-up shotgun lipidomics by higher energy collisional dissociation on LTQ Orbitrap mass spectrometers.Anal. Chem. 2011; 83: 5480-5487Crossref PubMed Scopus (102) Google Scholar) at the normalized collision energy (nCE) of 20% for native ecdysteroids and 40% for their Girard derivatives. Aliquots (10 µl) of the stock solution of ecdysone (5 mM in neat methanol) were spiked into 1 ml of 70% methanol (reaction temperature 50°C), 70% ethanol (70°C), and 70% isopropanol (85°C) in water. Then 50 μl of glacial acetic acid and 50 mg of the Girard reagent were added, and the mixture was incubated at the specified temperature. After certain time intervals, 5 μl aliquots were withdrawn and neutralized with 95 μl of methanol with 1% NH4OH. Then 10 μl aliquot of the neutralized sample was further diluted with 85 μl of water, and 5 μl of 0.5 μM muristerone A (internal standard) was spiked in. Of this mixture, 5 μl was injected into LC-MS system. Kinetics of the ecdysone derivatization was monitored by LC-MS/MS on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with Agilent 1200 LC system and operated with 60 min gradient elution program as described above. TSQ Vantage was equipped with a H-ESI ion source operated under spray voltage of 2.7 kV, sheath gas pressure of 5 psi, auxiliary gas set at 2 arbitrary units, vaporizer temperature at 50°C, and transfer capillary temperature at 200°C. The sample was loaded, concentrated, and cleaned up at the trap column during first 10 min and then eluted into the analytical column. All samples were analyzed in duplicates. Dh-G(p)E was quantified in MRM mode by monitoring pyridine neutral loss 580.4→501.4 (collision energy of 31 eV, S-lens at 215 V). Muristerone A (2.5 nM) spiked into samples as the internal standard was detected by monitoring the mass transition 497.4→425.4 (collision energy of 16 eV, S-lens at 145 V). Quantification was performed by the QualBrowser program from Xcalibur 2.2 software; chromatographic peaks were integrated using Genesis algorithm with five-point Gaussian smoothing. Preparative quantities of the product of ecdysone derivatization with Girard P were purified using two-step cleanup. First, Oasis MCX (6 cc/150 mg) cartridge from Waters was washed with 3 ml of methanol and then 3 ml of water. The reaction mixture was loaded in 15% aqueous methanol, and the cartridge was washed with 5 ml of 0.1 M aqueous hydrochloric acid. The derivatization product was eluted with 2 ml of 5% NH4OH in methanol and dried in a vacuum centrifuge. Then the sample was redissolved in 15% methanol, loaded on prewashed Oasis HLB (6 cc/150 mg) cartridge, washed with water, eluted with 2 ml of neat methanol, and vacuum dried. For NMR analysis, 2 mg of ecdysone and its Girard P derivative (purified as described above) were dissolved in DMSO-d6 at the concentration of 14 mM and 5.5 mM, respectively, and then the samples were transferred into 5 mm NMR tubes. 1H, 13C, 1H-13C-HSQC, 1H-13C-HMBC, 2D-COSY, and 2D-NOESY NMR spectra were recorded at 300 K on a Bruker AVANCE III 600 MHz spectrometer equipped with a cryogenic 5 mm TCI-H/C/N triple-resonance probe with actively shielded z-gradient. Spectra were referenced to the residual DMSO signal at 2.50 ppm and were processed and analyzed with TopSpin v.2.1 software (Bruker). Drosophila melanogaster WT (Oregon-K) embryos were collected for 2 h on apple juice/agar plates, rinsed in PBS containing 0.05% Triton X-100, bleached by 50% sodium hypochlorite for 30 s, and rinsed with distilled water. Then, embryos were transferred to plates containing standard lab food consisting of malt, soy, cornmeal, and yeast (7Carvalho M. Schwudke D. Sampaio J.L. Palm W. Riezman I. Dey G. Gupta G.D. Mayor S. Riezman H. Shevchenko A. et al.Survival strategies of a sterol auxotroph.Development. 2010; 137: 3675-3685Crossref PubMed Scopus (105) Google Scholar). Animals at the pupae stage were collected. Frozen animals were homogenized with a plastic pestle and extracted overnight with 1 ml of methanol at 4°C. After 5 min centrifuging at 14,500 rpm on a Mini-Spin centrifuge from Eppendorf (Hamburg, Germany), the supernatant was collected and further dried in a vacuum centrifuge. The pellet was twice reextracted with 1 ml of methanol for 30 min, which was combined and cleaned up by solid-phase extraction on a HLB Oasis cartridge as described (20Li Y. Warren J.T. Boysen G. Gilbert L.I. Gold A. Sangaiah R. Ball L.M. Swenberg J.A. Profiling of ecdysteroids in complex biological samples using liquid chromatography/ion trap mass spectrometry.Rapid Commun. Mass Spectrom. 2006; 20: 185-192Crossref PubMed Scopus (17) Google Scholar). The collected eluate was dried down and redissolved in 100 μl of 15% methanol prior to derivatization with Girard T (85°C, 4 h); the reaction mixture was cleaned up on an HLB Oasis cartridge. Endogenous ecdysteroids were detected by LC-MS/MS as described above; however, the injection volume was increased to 30 μl. MRM transitions and detection settings are provided in supplementary Table IV. Preliminary experiments performed under conventional reaction conditions (25Karu K. Hornshaw M. Woffendin G. Bodin K. Hamberg M. Alvelius G. Sjovall J. Turton J. Wang Y. Griffiths W.J. Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols.J. Lipid Res. 2007; 48: 976-987Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) (overnight incubation in 5% acetic acid in methanol; ambient temperature; starting reagent concentration of 0.3 mM) showed that the 6-ketone group of ecdysone was much less reactive toward hydrazides compared with 3-ketosterols. The Girard hydrazone was only produced at elevated reaction temperature; however we noticed that it was undergoing a rapid loss of water (Fig. 1). Therefore, we first investigated whether water loss occurred via eliminating a hydroxyl group from one or in parallel from several positions at the ecdysone backbone. We purified about 2 mg of dh-G(p)E by solid phase cleanup of the reaction mixture and analyzed it by NMR, which suggested that the specific loss of the 14-hydroxyl group yielded a double bond between C14-C15 (supplementary Fig. II), while other hydroxyl groups remained intact. Site-specific loss of the hydroxyl group corroborated LC-MS/MS analyses that revealed no alternative products that might differ by their retention times (see below). The control experiment performed without adding G(p) demonstrated that even at 85°C, ecdysone largely remained intact, suggesting that water loss occurred from or in parallel with the formation of G(p)E, rather than from ecdysone itself. Mass spectrum acquired by direct infusion of the reaction mixture purified by solid-phase extraction was dominated by the abundant peak of dh-G(p)E, while peaks corresponding to native E and G(p)E were minor (Fig. 2A). Therefore, we ventured to determine whether the yield of dh-G(p)E could be improved by varying the reaction time and temperature. Aliquots withdrawn from the reaction mixture at specified time points were cleaned up on a SPE cartridge and, after adding muristerone A (internal standard), were analyzed by LC-MS/MS. While at 50°C (conventional temperature for Girard derivatization) and 70°C the product yield was steadily increasing with time, at 85°C the abundance of dh-G(p)E peaked at the time point of around 4 h and then decreased, presumably due to progressing water losses and/or product decomposition (Fig. 2B). A separate MRM-based quantification experiment showed that only about 40% of ecdysone reacted by this time (data not shown); however, the abundance of dh-G(p)E exceeded the total abundance of all forms related to ecdysone (including molecular adducts) by more than 5-fold. These reaction conditions were subsequently used for detecting endogenous ecdysteroids. Next we derivatized ecdysone with a series of Girard reagents: G(p), G(t), and G(c). “Classic” Girard reagents G(p) and G(t) bear positively charged end groups (24Wheeler O.H. The Girard reagents.J. Chem. Educ. 1968; 45: 435-437Crossref Scopus (54) Google Scholar), whereas G(c) is better suited for negative mode analyses, which we thought might provide the analytical advantage of having alternative fragmentation pathways. Even at the low (<10 eV) collision energy, protonated molecular ions ([M+H]+) of ecdysteroids underwent successive loss of multiple (up to five, in the case of muristerone A) water molecules (21Wang Y.H. Avula B. Jadhav A.N. Smillie T.J. Khan I.A. Structural characterization and identification of ecdysteroids from Sida rhombifolia L. in positive electrospray ionization by tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2008; 22: 2413-2422Crossref PubMed Scopus (19) Google Scholar) (Figs. 3 and 4A). These relatively common fragmentation pathways did not contribute to higher detection specificity and compromised sensitivity of the analyses. At higher collision energy, we detected fragments produced by cleaving the bonds between C20-C22 (in E), C22-C23 (only observed in sterols with vicinal hydroxyls at C20 and C22, such as MA and 20H), and C17-C20 (8Blais C. Blasco T. Maria A. Dauphin-Villemant C. Lafont R. Characterization of ecdysteroids in Drosophila melanogaster by enzyme immunoassay and nano-liquid chromatography-tandem mass spectrometry.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2010; 878: 925-932Crossref PubMed Scopus (20) Google Scholar). In turn, these fragments also underwent successive water losses, yielding a series of relatively low abundant products (Fig. 3). In contrast, dh-G(p)E underwent rapid intramolecular cyclization accompanied by neutral loss of pyridine, either alone (Δm = 79.0; product m/z 501.3316) or together with concomitant loss of CO (Δm = 107.0; m/z 473.3371) (Fig. 4), similar to Girard hydrazones of 3-ketosterols (31Griffiths W.J. Tandem mass spectrometry in the study of fatty acids, bile acids, and steroids.Mass Spectrom. Rev. 2003; 22: 81-152Crossref PubMed Scopus (220) Google Scholar). CID MS3 experiments performed at the linear ion trap of a LTQ Orbitrap tandem mass spectrometer suggested that these were parallel (rather than successive) pathways because they produced nonoverlapping series of fragment ions (data not shown). Each of these two major fragments underwent up to two successive losses of hydroxyl groups from their hydrocarbon chains. Independently, the entire chain was split off by cleaving the C17-C20 bond (Fig. 5). At the same time, vicinal hydroxyls at the A-ring (positions C2 and C3) remained intact.Fig. 4Tandem mass spectra acquired from the dehydrated hydrazones of ecdysone and various Girard reagents. A: Native E. B: dh-G(p)E. C: dh-G(t)E. D: dh-G(c)E. MS/MS spectra in A–C were acquired in positive ion mode and in D in negative ion mode. Fragments designated with “-Py”, “-TMA,” and “-CO2” are products of neutral loss of, respectively, pyridine, TMA, and carbon dioxide from corresponding molecular cations of dehydrated Girard hydrazones. Collision energies were 23, 46, 46, and 39 eV, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Fragmentation pathways of dh-G(p)E in positive mode. Experimental m/z are from the spectrum in Fig. 4D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fragmentation of dh-G(t)E proceeded in the similar way. As expected, TMA neutral loss was a major pathway that produced the same fragments as dh-G(p)E (Fig. 4). We therefore reasoned that ecdysteroids could be detected by monitoring highly specific and abundant products of neutral loss of pyridine or TMA from their dehydrated precursors. In parallel, it should also be possible to monitor the fragments specific for the dehydroxylated at C14 position, but otherwise intact, sterol ring backbone. At the same time, we observed that no sterol core cleavage fragments or fragments of the side chain were sufficiently abundant for designing sensitive MRMs. For comparison, we derivatized E with G(c) and checked how dh-G(c)E was fragmenting in negative ion mode. Similar to G(p) and G(t) hydrazones in positive mode, the MS/MS spectrum was dominated by neutral loss of the end (here, carboxyl) group of Girard reagent accompanied by creating intramolecular cycle; however, there was no parallel pathway with further loss of CO (Fig. 6), and the fragments abundances were low. Altogether, derivatization with G(c) did not increase the detection sensitivity compared with native ecdysone, and it was not employed in further work. We then applied G(p) derivatization to seven individual ecdysteroids and observed both similar reaction efficiency and similar pathways of MS/MS fragmentation (supplementary Fig. II and supplementary Table I). Overall, the derivatization efficiency was similar for G(p) and G(t). However, G(t) derivatives were eluting about 30 s earlier than G(p) derivatives and offered slightly (on average, less than 2-fold) better sensitivity of MRM detection. Therefore, in our further work with endogenous hormones, we employed G(t). We next applied Girard derivatization for detecting endogenous ecdysteroids in Drosophila pupae. Ecdysteroids were enriched by extracting homogenized pupae with cold methanol. The total extract was treated with G(t) for 4 h at 85°C, cleaned up on the SPE cartridge, and analyzed by LC-MS/MS (Fig. 7). Dehydrated hydrazones of each expected ecdysteroid were monitored using MRM transitions relying upon the two major fragmentation pathways (Fig. 5): neutral loss of TMA was common to all G(t) derivatives; another complementary transition followed up a subsequent neutral loss of the hydrocarbon chains upon C17-C20 cleavage. By monitoring six transitions in parallel, we achieved confident detection of three major Drosophila ecdysteroids: E, 20H, and makisterone A at the endogenous level. In general, we were able to detect as low as 10 pg of ecdysteroids because of higher ionization capacity of corresponding dh-G(t) derivatives and reduced chemical noise in the monitored MRM transitions. Ecdysteroids are asymmetrical ketones and, upon treatment with Girard reagents, produced a mixture of E/Z hydrazones that were detected as pairs of chromatographically resolved peaks, which further supported their identification. Note that endogenous makisterone A (Fig. 7E, F) has an epimer, which may be detected as an additional chromatographic peak. Taken together, we demonstrated that Girard derivatization enhanced both the sensitivity and specificity of ecdysteroids detection by the method of MRM and that the endogenous ecdysteroids could be determined in the extract from a single Drosophila pupa or from 5–10 animals collected at the late larva stage. Girard derivatization is an established method for quantitative LC-MS profiling of mammalian oxysterols that have been enzymatically converted into 3-ketosterols. Our work further extended this “charge-tagging” approach to ecdysteroids. In contrast to 3-ketosterols, derivatization of the 6-ketone group in ecdysteroids was accompanied by facile loss of the 14-hydroxyl group, yielding an “additional” C14-C15 double bond. During MS/MS fragmentation, dh-G(t) and dh-G(p) ecdysteroids expectantly lost TMA and pyridine moieties, respectively, and a hydrocarbon side chain, while the steroid core remained intact. Together, these structure-specific neutral losses allowed us to design two complementary MRM transitions for each ecdysteroid and to recognize endogenous molecules in crude, lipid-rich extracts from larvae and pupae.

The selective uptake of compounds into specific cells of interest is a major objective in cell biology and drug delivery. By incorporation of a novel, thermostable azobenzene moiety we generated peptides that can be switched optically between an inactive state and an active, cell-penetrating state with excellent spatio-temporal control.

Lipoteichoic acid (LTA) is a major immunostimulatory molecule in the cell wall of Gram-positive bacteria. Adhesion of LTA to a polystyrene surface drastically increased its immunostimulatory potency in human whole blood in comparison to soluble LTA, although only 1% of the LTA had bound, as determined using rhodamine-labelled LTA. The release of the proinflammatory cytokines IL-1beta, TNF and IL-6 and the chemokines IL-8 and G-CSF was increased 2- to 10-fold, but IL-10 release was unaltered. This presentation effect was not shared by lipopolysaccharide (LPS) or other toll-like receptor 2 agonists and was less pronounced in polypropylene vessels. LTA did not induce cytokine release in silicone-coated borosilicate vessels, but covalent coupling of LTA to polystyrene beads restored cytokine induction in these vessels, indicating that presentation of LTA on a surface is in fact essential for its immunostimulatory potency. This novel aspect of presentation as a factor in the recognition of LTA may reflect the physiological situation in the bacterial cell wall, where LTA is anchored in the bacterial membrane and projects through the peptidoglycan. In practical terms, contamination of medical devices with components of Gram-positive bacteria may pose an underestimated inflammatory risk.

For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

The genetic integrity of each organism depends on the faithful segregation of its genome during mitosis. To meet this challenge, a cellular surveillance mechanism, termed the spindle assembly checkpoint (SAC), evolved that monitors the correct attachment of chromosomes and blocks progression through mitosis if corrections are needed. While the central role of the SAC for genome integrity is well established, its functional dissection has been hampered by the limited availability of appropriate small molecule inhibitors. Using a fluorescence polarization-based screen, we identify Mad2 inhibitor-1 (M2I-1), the first small molecule inhibitor targeting the binding of Mad2 to Cdc20, an essential protein-protein interaction (PPI) within the SAC. Based on computational and biochemical analyses, we propose that M2I-1 disturbs conformational dynamics of Mad2 critical for complex formation with Cdc20. Cellular studies revealed that M2I-1 weakens the SAC response, indicating that the compound might be active in cells. Thus, our study identifies the SAC specific complex formation between Mad2 and Cdc20 as a protein-protein interaction that can be targeted by small molecules.

Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.

Bile salts such as cholate are steroid compounds occurring ubiquitously in the environment through excretion by animals. Cholate degradation by Pseudomonas sp. strain Chol1 is initiated by A-ring oxidation and β-oxidation of the acyl side chain. A transposon mutant of strain Chol1 was isolated that could not grow with cholate, but transformed it into several steroid compounds accumulating in culture supernatants. The main product was identified as (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). A further compound was identified as 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). The structures of DHOCTO and THOCDO indicate that they are intermediates of the β-oxidation of the acyl side chain. The interrupted gene was named skt and had similarities to the 3-ketoacyl-CoA thiolase domain of the eukaryotic sterol carrier protein SCP-x. An skt mutant grew with intermediates of cholate degradation, from which the acyl side chain had been partly or completely removed. Growth with cholate was restored by an intact skt copy on a plasmid. These results strongly suggest that skt encodes a β-ketothiolase responsible for the cleavage of acetyl-CoA from the acyl side chain of cholate. Sequence comparisons revealed that other steroid-degrading bacteria such as Comamonas testosteroni contain genes encoding proteins very similar to Skt, suggesting a widespread role of this enzyme in bacterial steroid degradation.

Several zinc finger proteins have been discovered recently that bind specifically to double-stranded RNA. These include the mammalian JAZ and wig proteins, and the seven-zinc finger protein ZFa from Xenopus laevis. We have determined the solution structure of a 127 residue fragment of ZFa, which consists of two zinc finger domains connected by a linker that remains unstructured in the free protein in solution. The first zinc finger consists of a three-stranded beta-sheet and three helices, while the second finger contains only a two-stranded sheet and two helices. The common structures of the core regions of the two fingers are superimposable. Each finger has a highly electropositive surface that maps to a helix-kink-helix motif. There is no evidence for interactions between the two fingers, consistent with the length (24 residues) and unstructured nature of the intervening linker. Comparison with a number of other proteins shows similarities in the topology and arrangement of secondary structure elements with canonical DNA-binding zinc fingers, with protein interaction motifs such as FOG zinc fingers, and with other DNA-binding and RNA-binding proteins that do not contain zinc. However, in none of these cases does the alignment of these structures with the ZFa zinc fingers produce a consistent picture of a plausible RNA-binding interface. We conclude that the ZFa zinc fingers represent a new motif for the binding of double-stranded RNA.

Polypropylene glycol (PPG) is commonly added to bacterial cultures to avoid foaming. However, lipoteichoic acid (LTA) from bacteria grown with PPG lacked cytokine-inducing potency in human blood. We tested the blocking efficacy of several glycols on the cytokine response to staphylococcal LTA in human blood. PPG 1200 was the most potent inhibitor tested, shown for TNF, IL-1beta, IL-6, IL-8, IL-10 and TGF-beta induction, and displayed no cytotoxic effects. TNF induction by Staphylococcus aureus or by Toll-like receptor (TLR)2 agonists (di- and triacylated lipopeptides and LTA) was also inhibited by PPG 1200, but not that induced by Escherichia coli or TLR4 agonists. In flow cytometric studies, PPG-carrying nanobeads bound more rhodamine-labeled LTA than those with glycerol. Additionally, the methyl group peak in the (1)H-NMR of LTA shifted after incubation with increasing PPG 1200 concentrations. Sequential incubation of polystyrene plates with LTA, then PPG 1200 and then blood, with washing steps in between, showed that LTA-induced TNF release was inhibited. But when PPG 1200 was pre-incubated with blood that was washed before LTA was added, TNF induction was not repressed, demonstrating that PPG binds LTA and not cellular structures. In summary, PPG 1200 is a novel inhibitor of cytokine induction by TLR2 agonists, which interferes directly with the ligands.

The transmembrane protein YuaF from B. subtilis is a member of the NfeD-like clan with a potential role in maintaining membrane integrity during conditions of cellular stress. nfeD-genes are primarily found in highly conserved operon structures together with the gene of another membrane protein belonging to the SPFH superfamily, in this case YuaG. This strongly suggests a functional if not physical interaction between YuaF and YuaG. Secondary structure predictions of NfeD proteins that accompany SPFH proteins all indicate a high content of β-sheets in the C-terminal domains indicating a conserved core structure despite very low homology at the level of primary structure. Here we report the high-resolution solution structure of YuaF’s soluble C-terminal domain derived from NMR data (sYuaF, residues 97–174 of full-length YuaF). Full backbone and side chain assignments of sYuaF were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from 3D NOESY spectra collected at 600 MHz and 800 MHz, together with φ, ψ, and χ1 torsion angle restraints based on the analysis of 1HN, 15N, 1Hα, 13Cα, 13CO, and 13Cβ chemical shifts, and HNHA, HNHB and HACAHB-COSY spectra. Structures were calculated using CYANA 2.0 and refined in AMBER 8. sYuaF is composed of an extended N-terminal α-helix and a β-barrel formed by five β-strands. This β-sheet core structure is well known from the diverse class of OB-fold proteins and can also be found in the distantly related NfeD protein Ph0471 from the archaeon P. horikoshii. Despite significant differences of their amino acid sequences the structural homology of these proteins suggests a conserved function of SPFH-associated NfeD proteins.

The first phytochemical investigation of the flowers of Millettia dura resulted in the isolation of seven isoflavones, a flavonol and a chalcone. Eleven isoflavones and a flavonol isolated from various plant parts from this plant were tested for cytotoxicity against a panel of cell lines, and six of these showed good activity with IC50 values of 6-14 μM. Durmillone was the most active with IC50 values of 6.6 μM against A549 adenocarcinomic human alveolar basal epithelial cancer cell line with low cytotoxicity against the non-cancerous cell lines BEAS-2B (IC50 = 58.4 μM), LO2 hepatocytes (IC50 78.7 μM) and CCD19Lu fibroblasts (IC50 >100 μM).

The Na + -translocating NADH:ubiquinone oxidoreductase (Na + -NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA–NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA 1–377 was solved in space groups C 222 1 and P 2 1 by SAD phasing and molecular replacement at 1.9 and 2.1 Å resolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Å resolution.

Bacterial pathogens are influenced by signaling molecules including the catecholamines adrenaline and noradrenaline which are host-derived hormones and neurotransmitters. Adrenaline and noradrenaline modulate growth, motility and virulence of bacteria. We show that adrenaline is converted by the pathogen Vibrio cholerae to adrenochrome in the course of respiration, and demonstrate that superoxide produced by the respiratory, Na+ − translocating NADH:quinone oxidoreductase (NQR) acts as electron acceptor in the oxidative conversion of adrenaline to adrenochrome. Adrenochrome stimulates growth of V. cholerae, and triggers specific responses in V. cholerae and in immune cells. We performed a quantitative proteome analysis of V. cholerae grown in minimal medium with glucose as carbon source without catecholamines, or with adrenaline, noradrenaline or adrenochrome. Significant regulation of proteins participating in iron transport and iron homeostasis, in energy metabolism, and in signaling was observed upon exposure to adrenaline, noradrenaline or adrenochrome. On the host side, adrenochrome inhibited lipopolysaccharide-triggered formation of TNF-α by THP-1 monocytes, though to a lesser extent than adrenaline. It is proposed that adrenochrome produced from adrenaline by respiring V. cholerae functions as effector molecule in pathogen-host interaction.

The immunoglobulin superfamily protein neurolin plays a central role during differentiation and development of retina ganglion cells in goldfish. As shown in earlier work, blockage of the second immunoglobulin domain (Ig2) of neurolin with domain-specific antibodies causes severe pathfinding defects of growing axons in the retina. Thus Ig2 of neurolin was identified as the critical domain for axon guidance. In the present study we have developed a protocol for expression and purification of neurolin-Ig2 suitable for structure analysis, functional studies and ligand identification. Neurolin was expressed in Rosettagami and Origami strains of Escherichia coli which is deficient in glutathione- and thioredoxin reductase facilitating proper formation of the disulfide bond in the cytoplasm. The protein was purified via an N-terminal His(6)-tag by Ni(2+) affinity and size exclusion chromatography. After purification the His(6)-tag was cut-off without loss of solubility. Analytical size exclusion chromatography revealed an apparent molecular mass for neurolin-Ig2 in agreement with a non-covalent homodimer. Analysis of CD and FTIR spectra gave a secondary structure content typical for Ig domains.

Finding the right fit: Herein, we report on the development of novel steric probes and present initial insights into their interplay with DNA polymerases. Our findings provide experimental evidence for varied enzyme-substrate interactions that might account for the varied selectivity previously observed.

On the tracks of a DNA polymerase: NMR provides insights into DNA synthesis in a virtually label-free manner and under close-to-physiological conditions. Through the monitoring of the chemical-shift changes of multiple 13C-methyl methionine residues we found unique recognition states for canonical and noncanonical cases, thus indicating enzymatic cycling through distinct paths.

Abstract The complex formation of the following diazaperylene ligands (L) 1,12‐diazaperylene 1 , 1,1′‐bisisoquinoline 2 , 2,11‐disubstituted 1,12‐diazaperylenes (alkyl = methyl, ethyl, isopropyl, 3 , 5 , 7 ), 3,3′‐disubstituted 1,1′‐bisisoquinoline (alkyl = methyl, ethyl, isopropyl, 4 , 6 , 8 and with R = phenyl, 11 and with pyridine 12 ), and the 5,8‐dimethoxy‐substituted diazaperylene 9 , 6,6′‐dimethoxy‐substituted bisisoquinoline 10 with AgBF4 was investigated. Collision‐induced dissociation measurements were used to evaluate the relative stabilities of the ligands themselves and for the [1:1] + complexes as well as for the homoleptic and heteroleptic silver [1:2] + complexes in the gas phase. This method is very useful in rapid screening of the stabilities of new complexes in the gas phase. The influence of the spatial arrangement of the ligands and the type of substituents employed for the complexation were examined. The effect of the preorganization of the diazaperylene on the threshold activation voltages and thus of the relative binding energies of the different complexes are discussed. Density functional theory calculations were used to calculate the optimized structures of the silver complexes and compared with the stabilities of the complexes in the gas phase for the first time.

Hepcidin-25 was identified as the main iron regulator in the human body by binding to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as ATCUN (amino terminal Cu(II)- and Ni(II)- binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the formed complex of hepcidin-25 with copper could reveal insights into its biological role. The present work is mainly focused on the study of the metal-bound form of hepcidin-25, which could be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25, which was achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry MS/MS, high resolution mass spectrometry HRMS) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a 3D model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.

Abstract Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance and haemostasis, but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, focal adhesion assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of focal adhesion sites, with the cytoplasmic tails of the integrin β1 and β3 subunits. The binding interface resides in paxillin’s LIM3 domain, where based on the NMR structure and functional analyses a flexible, seven amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin β3 compromises cell adhesion and motility. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin β3 function.

The emergence of resistance to the existing TB drugs necessitated a search for new anti-mycobacterial compounds from Z. leprieurii root bark, which is used locally for the treatment of tuberculosis (TB) in Uganda. Two new compounds: (E)-N-isobutyl-3-(4-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylamide (1) and 4-(5,5a,6,8,8a,9-hexahydrofuro[3′,4′:6,7]-naphtha[2,3-d][1,3]dioxol-5-yl)-2-methoxyphenol (2), were isolated from the dichloromethane (DCM) extract. In addition, sixteen known compounds were isolated from the DCM and methanol extracts, with two of them, N-isobutylcinnamamide (9) and (E)-3-(4-hydroxy-3-methoxyphenyl)-N-isobutylacrylamide (14), being reported from a natural source for the first time. The structures of the isolates were elucidated by a combination of spectroscopic techniques. The isolated compounds were subjected to anti-mycobacterial activity testing using the microplate Alamar blue assay (MABA). Five compounds, 10 (MIC, 0.98 μg/mL), 11 (MIC, 7.82 μg/mL), 12 (MIC, 1.95 μg/mL), 13 (MIC, 3.91 μg/mL), and 14 (MIC, 3.91 μg/mL) exhibited significant anti-mycobacterial activities against the susceptible (H37Rv) strain and could provide vital templates for developing new and highly effective TB drugs. Also, compounds 4 (MIC, 62.50 μg/mL), 7 (MIC, 62.50 μg/mL) and 16 (MIC, 62.50 μg/mL) exhibited moderate activity against H37Rv.

We present a divergent radical strategy for the fluorination of phenylacetic acid derivatives through N–F bond activation of Selectfluor® with 4-(dimethylamino)pyridine. Comprehensive reaction investigation revealed the critical role of reaction media on selectivity. In presence of water, decarboxylative fluorination through a single electron oxidation is dominant. Non-aqueous conditions result in the clean formation of α-fluoro-α-arylcarboxylic acids through hydrogen atom transfer.

No abstract available

Bile salts such as cholate are steroid compounds from the digestive tracts of vertebrates, which enter the environment upon excretion, e.g., in manure. Environmental bacteria degrade bile salts aerobically via two pathway variants involving intermediates with Δ1,4- or Δ4,6-3-keto-structures of the steroid skeleton. Recent studies indicated that degradation of bile salts via Δ4,6-3-keto intermediates in Sphingobium sp. strain Chol11 proceeds via 9,10-seco cleavage of the steroid skeleton. For further elucidation, the presumptive product of this cleavage, 3,12β-dihydroxy-9,10-seco-androsta-1,3,5(10),6-tetraene-9,17-dione (DHSATD), was provided to strain Chol11 in a co-culture approach with Pseudomonas stutzeri Chol1 and as purified substrate. Strain Chol11 converted DHSATD to the so far unknown compound 4-methyl-3-deoxy-1,9,12-trihydroxyestra-1,3,5(10)7-tetraene-6,17-dione (MDTETD), presumably in a side reaction involving an unusual ring closure. MDTETD was neither degraded by strains Chol1 and Chol11 nor in enrichment cultures. Functional transcriptome profiling of zebrafish embryos after exposure to MDTETD identified a significant overrepresentation of genes linked to hormone responses. In both pathway variants, steroid degradation intermediates transiently accumulate in supernatants of laboratory cultures. Soil slurry experiments indicated that bacteria using both pathway variants were active and also released their respective intermediates into the environment. This instance could enable the formation of recalcitrant steroid metabolites by interspecies cross-feeding in agricultural soils.

The inside cover picture shows the KlenTaq DNA polymerase in complex with DNA and a matching nucleoside triphosphate. H. M. Möller et al. (see article on p. 635 ff.) have identified unique recognition states of this enzyme by monitoring the NMR chemical-shift perturbations of 13C-labeled methionine methyl groups upon substrate binding. (The coordinates of the 3D structure were taken from PDB ID 3KTQ.)

Neurolin is a member of the superfamily of immunoglobulin-like cell surface receptors. It is essential during neuronal development in the model organism Carassius auratus (goldfish) and involved in the guidance of the growing axon. Among the five extracellular immunoglobulin (Ig) domains, the second Ig domain is crucial for axon pathfinding. In the present study, we report the NMR assignment and secondary structure prediction of the second Ig domain of neurolin.

Today’s scoring functions are one of the main reasons that state-of-the-art protein-ligand dockings fail in about 20 % to 40 % of the targets due to the sometimes severe approximations they make. However these approximations are necessary for performance reasons. One possibility to overcome these problems is the inclusion of additional, preferably experimental information in the docking process. Especially ligand-based NMR experiments that are far less demanding than the solution of the whole complex structure are helpful. Here we present the inclusion of three different types of NMR-data into the ChemPLP [1] scoring function of our docking tool PLANTS [2]. First, STD and intraligand trNOE spectra were used to obtain distant constraints between ligand and protein atoms. This approach proved beneficial for the docking of larger peptide ligands i. e. the epitope of MUC-1 glycoprotein to the SM3 antibody [3]. In the second part the usefulness of INPHARMA data [4,5] is shown by combinig a score, evaluating the agreement between simulated and measured INPHARMA spectra, with the PLANTS ChemPLP scoring function. First results from rescoring after local optimization of the poses and full docking experiments are shown.

Despite the fact that many different programs have been developed in the field of NMR-based determination, the 3D determination of a large protein is still very time consuming and needs a lot of manual intervention. Therefore, it is highly desired to automate as many of the steps peak picking, assignment, and structure calculation as possible in a self-consistent manner. To be successful, such programs have to be very flexible to benefit from different types of constraints and robust in dealing with ambiguities and noise in the spectra.

Abstract The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic Synopsis Nanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

Eukaryotes have proteins anchored to the cells’ plasma membrane via the glycolipid anchor glycosylphosphatidylinositol (GPIs). GPI anchored proteins (GPI-APs) can play a role in the protection, regulation and activation of cells via protein-protein interactions. Studies of GPIs and GPI-APs are hindered by the difficulty of isolating and producing these complex molecules in pure forms. Herein, we apply a semi-synthetic method to synthesize well-defined GPI-APs by combining protein expression and chemo-selective attachment of synthetic GPI to proteins.