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Martine Biard-Piechaczyk

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DOI: 10.4161/auto.19496
2012
Cited 3,113 times
Guidelines for the use and interpretation of assays for monitoring autophagy
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
DOI: 10.4161/auto.5338
2008
Cited 1,663 times
Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
DOI: 10.1016/j.biochi.2007.08.014
2008
Cited 466 times
Regulation of macroautophagy by mTOR and Beclin 1 complexes
Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.
DOI: 10.1128/jvi.76.24.12813-12822.2002
2002
Cited 439 times
The Complementary Strand of the Human T-Cell Leukemia Virus Type 1 RNA Genome Encodes a bZIP Transcription Factor That Down-Regulates Viral Transcription
The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral trans-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.
DOI: 10.1371/journal.ppat.1002422
2011
Cited 175 times
IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network
Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.
DOI: 10.1182/blood-2002-03-0978
2003
Cited 159 times
Role of the intracellular domains of CXCR4 in SDF-1–mediated signaling
The CXCR4 chemokine receptor is a Gi protein–coupled receptor that triggers multiple intracellular signals in response to stromal cell-derived factor 1 (SDF-1), including calcium mobilization and p44/42 extracellular signal-regulated kinases (ERK1/2). Transduced signals lead to cell chemotaxis and are terminated through receptor internalization depending on phosphorylation of the C terminus part of CXCR4. Receptor endocytosis is also required for some receptors to stimulate ERK1/2 and to migrate through a chemokine gradient. In this study, we explored the role played by the 3 intracellular loops (ICL1-3) and the C terminus domain of CXCR4 in SDF-1–mediated signaling by using human embryonic kidney (HEK)–293 cells stably expressing wild-type or mutated forms of CXCR4. ICL3 of CXCR4 is specifically involved in Gi-dependent signals such as calcium mobilization and ERK activation, but does not trigger CXCR4 internalization after SDF-1 binding, indicating that ERK phosphorylation is independent of CXCR4 endocytosis. Surprisingly, ICL2, with or without the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for Gi signaling. However, ICL2 and ICL3, as well as the C terminus part of CXCR4, are needed to transduce SDF-1–mediated chemotaxis, suggesting that this event involves multiple activation pathways and/or cooperation of several cytoplasmic domains of CXCR4.
DOI: 10.1128/jvi.02174-14
2015
Cited 125 times
Autophagy Restricts HIV-1 Infection by Selectively Degrading Tat in CD4 <sup>+</sup> T Lymphocytes
ABSTRACT Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4 + T lymphocytes, a mechanism likely contributing to the loss of CD4 + T cells. In contrast, in productively infected CD4 + T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4 + T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread. IMPORTANCE Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4 + T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.
DOI: 10.1128/jvi.75.16.7637-7650.2001
2001
Cited 117 times
Binding of Human Immunodeficiency Virus Type 1 gp120 to CXCR4 Induces Mitochondrial Transmembrane Depolarization and Cytochrome<i>c</i>-Mediated Apoptosis Independently of Fas Signaling
Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.
DOI: 10.4161/auto.6880
2008
Cited 114 times
HIV-1 gp41 fusogenic function triggers autophagy in uninfected cells
AbstractCell-expressed HIV-1 envelope glycoproteins (gp120 and gp41, called Env) induce autophagy in uninfected CD4 T cells, leading to their apoptosis, a mechanism most likely contributing to immunodeficiency. The presence of CD4 and CXCR4 on target cells is required for this process, but Env-induced autophagy is independent of CD4 signaling. Here, we demonstrate that CXCR4-mediated signaling pathways are not directly involved in autophagy and cell death triggering. Indeed, cells stably expressing mutated forms of CXCR4, unable to transduce different Gi-dependent and -independent signals, still undergo autophagy and cell death after coculture with effector cells expressing Env. After gp120 binding to CD4 and CXCR4, the N terminus fusion peptide (FP) of gp41 is inserted into the target membrane, and gp41 adopts a trimeric extended pre-hairpin intermediate conformation, target of HIV fusion inhibitors such as T20 and C34, before formation of a stable six-helix bundle structure and cell-to-cell fusion. Interestingly, Env-mediated autophagy is triggered in both single cells (hemifusion) and syncytia (complete fusion), and prevented by T20 and C34. The gp41 fusion activity is responsible for Env-mediated autophagy since the Val2Glu mutation in the gp41 FP totally blocks this process. On the contrary, deletion of the C-terminal part of gp41 enhances Env-induced autophagy. These results underline the major role of gp41 in inducing autophagy in the uninfected cells and indicate that the entire process leading to HIV entry into target cells through binding of Env to its receptors, CD4 and CXCR4, is responsible for autophagy and death in the uninfected, bystander cells.
DOI: 10.1371/journal.pone.0005787
2009
Cited 114 times
Differential Role of Autophagy in CD4 T Cells and Macrophages during X4 and R5 HIV-1 Infection
HIV-1 can infect and replicate in both CD4 T cells and macrophages. In these cell types, HIV-1 entry is mediated by the binding of envelope glycoproteins (gp120 and gp41, Env) to the receptor CD4 and a coreceptor, principally CCR5 or CXCR4, depending on the viral strain (R5 or X4, respectively). Uninfected CD4 T cells undergo X4 Env-mediated autophagy, leading to their apoptosis, a mechanism now recognized as central to immunodeficiency.We demonstrate here that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, we observed two populations of autophagic cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection.Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.
DOI: 10.1006/viro.1999.0151
2000
Cited 104 times
Caspase-Dependent Apoptosis of Cells Expressing the Chemokine Receptor CXCR4 Is Induced by Cell Membrane-Associated Human Immunodeficiency Virus Type 1 Envelope Glycoprotein (gp120)
Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins interact with CD4 and chemokine receptors on T cells to deliver signals that trigger either activation, anergy, or apoptosis. However, the molecular mechanisms driving these responses remain poorly understood. In this study we demonstrate that apoptosis is induced upon HIV-1 envelope binding to the chemokine receptor CXCR4. Cells expressing a mutant form of CXCR4 with a C-terminal deletion were also sensitive to HIV-1 envelope-mediated apoptosis, indicating that the cytoplasmic tail of CXCR4 is not required to induce the apoptotic pathway. The specificity of this process was analyzed using several inhibitors of gp120-CD4–CXCR4 interaction. Monoclonal antibodies directed against the gp120-binding site on CD4 (ST4) and against CXCR4 (MAB173) prevented the apoptotic signal in a dose-dependent manner. The cell death program was also inhibited by SDF-1α, the natural ligand of CXCR4, and by suramin, a G protein inhibitor that binds with a high affinity to the V3 loop of HIV-1 gp120 envelope protein. These results highlight the role played by gp120-binding on CXCR4 to trigger programmed cell death. Next, we investigated the intracellular signal involved in gp120-induced apoptosis. This cell death program was insensitive to pertussis toxin and did not involve activation of the stress- and apoptosis-related MAP kinases p38MAPK and SAPK/JNK but was inhibited by a broad spectrum caspase inhibitor (z-VAD.fmk) and a relatively selective inhibitor of caspase 3 (z-DEVD.fmk). Altogether, our results demonstrate that HIV induces a caspase-dependent apoptotic signaling pathway through CXCR4.
DOI: 10.1186/1742-4690-1-12
2004
Cited 85 times
Apoptosis of uninfected cells induced by HIV envelope glycoproteins.
Apoptosis, or programmed cell death, is a key event in biologic homeostasis but is also involved in the pathogenesis of many human diseases including human immunodeficiency virus (HIV) infection. Although multiple mechanisms contribute to the gradual T cell decline that occurs in HIV-infected patients, programmed cell death of uninfected bystander T lymphocytes, including CD4+ and CD8+ T cells, is an important event leading to immunodeficiency. The HIV envelope glycoproteins (Env) play a crucial role in transducing this apoptotic signal after binding to its receptors, the CD4 molecule and a coreceptor, essentially CCR5 and CXCR4. Depending on Env presentation, the receptor involved and the complexity of target cell contact, apoptosis induction is related to death receptor and/or mitochondria-dependent pathways. This review summarizes current knowledge of Env-mediated cell death leading to T cell depletion and clinical complications and covers the sometimes conflicting studies that address the possible mechanisms of T cell death.
DOI: 10.1007/s00109-007-0173-6
2007
Cited 80 times
Involvement of autophagy in viral infections: antiviral function and subversion by viruses
Autophagy is a cellular process involved in the degradation and turn-over of long-lived proteins and organelles, which can be subjected to suppression or further induction in response to different stimuli. According to its essential role in cellular homeostasis, autophagy has been implicated in several pathologies including cancer, neurodegeneration and myopathies. More recently, autophagy has been described as a mechanism of both innate and adaptive immunity against intracellular bacteria and viruses. In this context, autophagy has been proposed as a protective mechanism against viral infection by degrading the pathogens into autolysosomes. This is strengthened by the fact that several proteins involved in interferon (IFN) signalling pathways are linked to autophagy regulation. However, several viruses have evolved strategies to divert IFN-mediated pathways and autophagy to their own benefit. This review provides an overview of the autophagic process and its involvement in the infection by different viral pathogens and of the connections existing between autophagy and proteins involved in IFN signalling pathways.
DOI: 10.1097/qad.0000000000000554
2015
Cited 53 times
HIV-1 viral infectivity factor interacts with microtubule-associated protein light chain 3 and inhibits autophagy
Objective: Autophagy, an important antiviral process triggered during HIV-1 entry by gp41-dependent membrane fusion, is repressed in infected CD4+ T cells by an unknown mechanism. The aim of this study was to identify the role of viral infectivity factor (Vif) in the autophagy blockade. Design/methods: To determine the role of Vif in autophagy inhibition, we used cell lines that express CD4 and CXCR4 and primary CD4+ T cells. Pull-down experiments, immunoprecipitation assays and computational analyses were performed to analyze the interaction between Vif and microtubule-associated protein light chain 3B (LC3B), a major autophagy component, in presence or absence of the antiviral host factor apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), after HIV-1 infection or ectopic expression of Vif. Autophagy was analyzed after infection by viruses expressing Vif (NL4.3) or not (NL4.3ΔVif), or after exogenous Vif expression. Results: We demonstrate that the C-terminal part of Vif interacts directly with LC3B, independently of the presence of APOBEC3G.Vif binds to pro-LC3 and autophagy-related protein 4-cleaved LC3 forms, and glycine 120, the amino acid conjugated to phosphatidylethanolamine on autophagosomes, is required. Importantly, we evidence that Vif inhibits autophagy during HIV-1 infection. Indeed, autophagy is detected in target cells infected by NL4.3ΔVif, but prevented in cells infected by NL4.3. Furthermore, autophagy triggered in NL4.3ΔVif-infected cells is inhibited when Vif is expressed in trans but is still active when target cells express a mutant of Vif that binds weakly to LC3B. Conclusion: Our study unveils that Vif inhibits autophagy independently of its action on APOBEC3G and, therefore, suggest a new function of this viral protein in restricting innate antiviral mechanisms.
DOI: 10.1186/s12977-015-0181-5
2015
Cited 49 times
The HDAC6/APOBEC3G complex regulates HIV-1 infectiveness by inducing Vif autophagic degradation
Human immunodeficiency virus type 1 (HIV-1) has evolved a complex strategy to overcome the immune barriers it encounters throughout an organism thanks to its viral infectivity factor (Vif), a key protein for HIV-1 infectivity and in vivo pathogenesis. Vif interacts with and promotes "apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3G" (A3G) ubiquitination and subsequent degradation by the proteasome, thus eluding A3G restriction activity against HIV-1.We found that cellular histone deacetylase 6 (HDAC6) directly interacts with A3G through its C-terminal BUZ domain (residues 841-1,215) to undergo a cellular co-distribution along microtubules and cytoplasm. The HDAC6/A3G complex occurs in the absence or presence of Vif, competes for Vif-mediated A3G degradation, and accounts for A3G steady-state expression level. In fact, HDAC6 directly interacts with and promotes Vif autophagic clearance, thanks to its C-terminal BUZ domain, a process requiring the deacetylase activity of HDAC6. HDAC6 degrades Vif without affecting the core binding factor β (CBF-β), a Vif-associated partner reported to be key for Vif- mediated A3G degradation. Thus HDAC6 antagonizes the proviral activity of Vif/CBF-β-associated complex by targeting Vif and stabilizing A3G. Finally, in cells producing virions, we observed a clear-cut correlation between the ability of HDAC6 to degrade Vif and to restore A3G expression, suggesting that HDAC6 controls the amount of Vif incorporated into nascent virions and the ability of HIV-1 particles of being infectious. This effect seems independent on the presence of A3G inside virions and on viral tropism.Our study identifies for the first time a new cellular complex, HDAC6/A3G, involved in the autophagic degradation of Vif, and suggests that HDAC6 represents a new antiviral factor capable of controlling HIV-1 infectiveness by counteracting Vif and its functions.
DOI: 10.1074/jbc.m408481200
2005
Cited 66 times
Identification of the Cytoplasmic Domains of CXCR4 Involved in Jak2 and STAT3 Phosphorylation
The chemokine SDF-1α transduces Gi-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a Gi-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1α binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr135 in the DRY motif at the N terminus of ICL2 nor the Tyr65 and Tyr76 in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1α binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling. The chemokine SDF-1α transduces Gi-dependent and -independent signals through CXCR4. Activation of Jak2/STAT3, a Gi-independent signaling pathway, which plays a major role in survival signals, is known to be activated after SDF-1α binding to CXCR4 but the domains of CXCR4 involved in this signaling remain unexplored. Using human embryonic kidney HEK-293 cells stably expressing wild-type or mutated forms of CXCR4, we demonstrated that STAT3 phosphorylation requires the N-terminal part of the third intracellular loop (ICL3) and the tyrosine 157 present at the end of the second intracellular loop (ICL2) of CXCR4. In contrast, neither the conserved Tyr135 in the DRY motif at the N terminus of ICL2 nor the Tyr65 and Tyr76 in the first intracellular loop (ICL1) are involved in this activation. ICL3, which does not contain any tyrosine residues, is needed to activate Jak2. These results demonstrate that two separate domains of CXCR4 are involved in Jak2/STAT3 signaling. The N-terminal part of ICL3 is needed to activate Jak2 after SDF-1α binding to CXCR4, leading to phosphorylation of only one cytoplasmic Tyr, present at the C terminus of ICL2, which triggers STAT3 activation. This work has profound implications for the understanding of CXCR4-transduced signaling. The seven transmembrane G protein-coupled receptor (GPCR) 1The abbreviations used are: GPCR, G protein-coupled receptor; SDF, stromal-derived factor 1; Ab, antibody; mAb, monoclonal antibody; HA, hemagglutinin; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; Jak, Janus kinase; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; HIV, human immunodeficiency virus; ICL, intracellular loop; STAT, signal transduction and activation of transcription. CXCR4 is expressed on many cell types and binds the CXC chemokine stromal-derived factor 1 (SDF-1α), also named CXCL12 (1Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1994) Google Scholar, 2Rollins B.J. Blood. 1997; 90: 909-928Crossref PubMed Google Scholar). SDF-1α/CXCR4 plays a role in embryonic development, regulation of cell proliferation, and migration (3Bleul C.C. Farzan M. Choe H. Parolin C. Clark-Lewis I. Sodroski J. Springer T.A. 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After binding to CXCR4, SDF-1α activates multiple signal transduction pathways including phosphorylation of focal adhesion components, extracellular signal-regulated kinases 1 and 2 (ERK1 and 2), phospholipase C-γ, protein kinase C, and phosphatidylinositol 3-kinase as well as calcium mobilization, activation of the nuclear factor κB (NFκB) and chemotaxis (15Ganju R.K. Brubaker S.A. Meyer J. Dutt P. Yang Y. Qin S. Newman W. Groopman J.E. J. Biol. Chem. 1998; 273: 23169-23175Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 16Wang J.F. Park I.W. Groopman J.E. Blood. 2000; 95: 2505-2513Crossref PubMed Google Scholar). Activation of the Janus kinase (Jak)/signal transduction and activation of transcription (STAT) pathway is also triggered after SDF-1α binding to CXCR4 in T, B, monocytic, and hematopoietic progenitor cell lines (17Soriano S.F. Hernanz-Falcon P. Rodriguez-Frade J.M. De Ana A.M. Garzon R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martinez A.C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar, 18Vila-Coro A.J. Rodriguez-Frade J.M. Martin De Ana A. Moreno-Ortiz M.C. Martinez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 19Zhang X.F. Wang J.F. Matczak E. Proper J.A. Groopman J.E. Blood. 2001; 97: 3342-3348Crossref PubMed Scopus (143) Google Scholar). Previous studies have shown that chemokine binding to its receptor leads to activation of Jak proteins (20Mellado M. Rodriguez-Frade J.M. Manes S. Martinez A.C. Annu. Rev. Immunol. 2001; 19: 397-421Crossref PubMed Scopus (301) Google Scholar). Activated Jaks in turn phosphorylate the chemokine receptor on intracellular tyrosines, providing docking sites for recruitment of STAT family proteins. After STAT phosphorylation and dimerization, these proteins translocate to the nucleus and mediate transcription of several cytokine-responsive genes (21Imada K. Leonard W.J. Mol. Immunol. 2000; 37: 1-11Crossref PubMed Scopus (486) Google Scholar, 22Jove R. Oncogene. 2000; 19: 2466-2467Crossref PubMed Scopus (54) Google Scholar, 23Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1493) Google Scholar). This pathway is independent of heterotrimeric Gi protein activation but Gαi protein association with GPCR is dependent on Jak activation (17Soriano S.F. Hernanz-Falcon P. Rodriguez-Frade J.M. De Ana A.M. Garzon R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martinez A.C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar). Stimulation of cells with chemokine has been shown to induce cell surface GPCR oligomerization that brings about the local aggregation of associated Jaks, resulting in their activation by transphosphorylation (20Mellado M. Rodriguez-Frade J.M. Manes S. Martinez A.C. Annu. Rev. Immunol. 2001; 19: 397-421Crossref PubMed Scopus (301) Google Scholar, 24Mellado M. Rodriguez-Frade J.M. Vila-Coro A.J. Fernandez S. Martin de Ana A. Jones D.R. Toran J.L. Martinez A.C. EMBO J. 2001; 20: 2497-2507Crossref PubMed Scopus (384) Google Scholar, 25Rodriguez-Frade J.M. Mellado M. Martinez A.C. Trends Immunol. 2001; 22: 612-617Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, several GPCRs are already expressed at the cell surface as dimers in the absence of ligand (26Fotiadis D. Liang Y. Filipek S. Saperstein D.A. Engel A. Palczewski K. Nature. 2003; 421: 127-128Crossref PubMed Scopus (661) Google Scholar, 27Gent J. Van Den Eijnden M. Van Kerkhof P. Strous G.J. Mol. Endocrinol. 2003; 17: 967-975Crossref PubMed Scopus (59) Google Scholar, 28Gent J. van Kerkhof P. Roza M. Bu G. Strous G.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9858-9863Crossref PubMed Scopus (160) Google Scholar, 29Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. 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FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar) demonstrated that SDF-1α binding to CXCR4 induces receptor dimerization, but recent data indicate that CXCR4 can also form constitutive dimers without ligand stimulation (33Babcock G.J. Farzan M. Sodroski J. J. Biol. Chem. 2003; 278: 3378-3385Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 34Toth P.T. Ren D. Miller R.J. J. Pharmacol. Exp. Ther. 2004; 310: 8-17Crossref PubMed Scopus (84) Google Scholar). Several members of Jak and STAT proteins are activated depending on the receptor/ligand pair and on the cell types. Activation of the Jak2/STAT3 pathway was described in several chemokine receptors such as CCR2b, CXCR4, CCR5, and platelet-activating factor receptor (PAFR) (18Vila-Coro A.J. Rodriguez-Frade J.M. Martin De Ana A. Moreno-Ortiz M.C. Martinez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar, 35Lukashova V. Asselin C. Krolewski J.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 2001; 276: 24113-24121Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 36Mellado M. Rodriguez-Frade J.M. Aragay A. del Real G. Martin A.M. Vila-Coro A.J. Serrano A. Mayor Jr., F. Martinez A.C. J. Immunol. 1998; 161: 805-813Crossref PubMed Google Scholar, 37Wong M. Fish E.N. J. Biol. Chem. 1998; 273: 309-314Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) and Jak2 is required for SDF-1α-induced activation of PI 3-kinase, tyrosine phosphorylation of multiple focal adhesion proteins and chemotaxis of hematopoietic progenitor cells (19Zhang X.F. Wang J.F. Matczak E. Proper J.A. Groopman J.E. Blood. 2001; 97: 3342-3348Crossref PubMed Scopus (143) Google Scholar). Other GPCRs such as luteinizing hormone, angiotensin II, PAR-1, and serotonin 5-HT2A receptors also activate Jak2 after ligand binding (38Ali M.S. Sayeski P.P. Dirksen L.B. Hayzer D.J. Marrero M.B. Bernstein K.E. J. Biol. 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In the same way, STAT3 is ubiquitously expressed and plays an important role in cell growth regulation, inflammation and early embryonic development (43Aaronson D.S. Horvath C.M. Science. 2002; 296: 1653-1655Crossref PubMed Scopus (1070) Google Scholar). This STAT protein is required for cell migration and was shown to be activated after SDF-1α binding to CXCR4 (17Soriano S.F. Hernanz-Falcon P. Rodriguez-Frade J.M. De Ana A.M. Garzon R. Carvalho-Pinto C. Vila-Coro A.J. Zaballos A. Balomenos D. Martinez A.C. Mellado M. J. Exp. Med. 2002; 196: 311-321Crossref PubMed Scopus (57) Google Scholar, 18Vila-Coro A.J. Rodriguez-Frade J.M. Martin De Ana A. Moreno-Ortiz M.C. Martinez A.C. Mellado M. FASEB J. 1999; 13: 1699-1710Crossref PubMed Scopus (443) Google Scholar). Activation of the Jak/STAT signal transduction pathway depends on specific cytoplasmic domains of receptors. These domains have been well characterized for several cytokine receptors such as the interleukin-6 signal transducer gp130 (44Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 45Haan C. Heinrich P.C. Behrmann I. Biochem. J. 2002; 361: 105-111Crossref PubMed Scopus (48) Google Scholar), the interleukin-2 receptor β chain (46Zhu M.H. Berry J.A. Russell S.M. Leonard W.J. J. Biol. Chem. 1998; 273: 10719-10725Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), the interferon receptor chain 2c (47Velichko S. Wagner T.C. Turkson J. Jove R. Croze E. J. Biol. Chem. 2002; 277: 35635-35641Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), the granulocyte-macrophage colony-stimulating factor (GM-CSF) (48Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar), the cMpl (49Gurney A.L. Wong S.C. Henzel W.J. de Sauvage F.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5292-5296Crossref PubMed Scopus (177) Google Scholar), and the erythropoietin receptor (50Klingmuller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar). Depending on the receptor, one or several tyrosine residues are phosphorylated during Jak/STAT activation (44Gerhartz C. Heesel B. Sasse J. Hemmann U. Landgraf C. Schneider-Mergener J. Horn F. Heinrich P.C. Graeve L. J. Biol. Chem. 1996; 271: 12991-12998Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 47Velichko S. Wagner T.C. Turkson J. Jove R. Croze E. J. Biol. Chem. 2002; 277: 35635-35641Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 49Gurney A.L. Wong S.C. Henzel W.J. de Sauvage F.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5292-5296Crossref PubMed Scopus (177) Google Scholar, 51Bahrenberg G. Behrmann I. Barthel A. Hekerman P. Heinrich P.C. Joost H.G. Becker W. Mol. Endocrinol. 2002; 16: 859-872Crossref PubMed Scopus (74) Google Scholar, 52Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In contrast, nearly no information is available about the cytoplasmic domains of GPCRs needed for Jak/STAT signaling. To our knowledge, the only data on this subject focus on CCR2b and the angiotensin II AT1 receptor (36Mellado M. Rodriguez-Frade J.M. Aragay A. del Real G. Martin A.M. Vila-Coro A.J. Serrano A. Mayor Jr., F. Martinez A.C. J. Immunol. 1998; 161: 805-813Crossref PubMed Google Scholar, 53Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) in which the DRY site (36Mellado M. Rodriguez-Frade J.M. Aragay A. del Real G. Martin A.M. Vila-Coro A.J. Serrano A. Mayor Jr., F. Martinez A.C. J. Immunol. 1998; 161: 805-813Crossref PubMed Google Scholar) and the YIPP motif in the carboxyl tail (53Ali H. Fisher I. Haribabu B. Richardson R.M. Snyderman R. J. Biol. Chem. 1997; 272: 11706-11709Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), respectively, are essential for Jak2 activation. In the present study, we analyzed the cytoplasmic domains and the intracellular tyrosines of CXCR4 involved in Jak2/STAT3 activation after SDF-1α binding. To address this question, we used previously constructed stable HEK-293 cells that express mutated forms of CXCR4 in which each intracellular loop (ICL) was replaced by a scrambled amino acid sequence of ICL1 that does not contain serine, threonine and tyrosine, or a CXCR4 form truncated at position 308 (54Roland J. Murphy B.J. Ahr B. Robert-Hebmann V. Delauzun V. Nye K.E. Devaux C. Biard-Piechaczyk M. Blood. 2003; 101: 399-406Crossref PubMed Scopus (144) Google Scholar). We also constructed stable HEK-293 cells that express point mutation CXCR4 receptors in which each intracellular tyrosine, Tyr65 (ICL1), Tyr76 (ICL1), Tyr135 (ICL2), or Tyr157 (ICL2) was replaced by a nonphosphorylatable amino acid and deleted forms of ICL3. We demonstrate here that SDF-1α-mediated Jak2/STAT3 signaling is transduced through the N terminus of ICL3 and Tyr157 of CXCR4. Materials—SDF-1α, polyclonal rabbit anti-SDF-1α antibody (Ab), and mouse monoclonal (mAb) anti-CXCR4 (MAB173) antibody were purchased from R & D Systems (R & D Systems Europe, Abington, United Kingdom). The anti-HA and Jak2 Abs were purchased from Upstate Cell Signaling Solutions (Euromedex, Mundolsheim, France). Anti-FLAG Ab M2 was purchased from Sigma-Aldrich. Anti-phosphotyrosine mAb (PY20) and anti-phosphorylated ERK1/2 Ab were purchased from Santa Cruz Biotechnology (Tebu-bio, Le Perray en Yvelines, France). Anti-P-STAT3 (Tyr705) and anti-STAT3 Abs were purchased from Cell Signaling Technology (Ozyme, Saint Quentin Yvelines, France). Fluorescein isothiocyanate (FITC) and peroxidase-labeled Fab′2 anti-mouse and anti-rabbit immunoglobulins were from Sigma-Aldrich. Bordetella pertussis toxin (PTX) and AG490 were purchased from Calbiochem (France Biochem, Meudon, France). The pRK5-Jak2 vector was kindly provided by J. Ihle, St. Jude Children's Research Hospital, Memphis, TN. Construction of the CXCR4 Mutants and Tagged HA CXCR4 —Point mutation mutants of CXCR4, in which tyrosines at positions 65, 76, 135, or 157 were replaced by alanine or phenylalanine, were constructed using GeneEditor in vitro site-directed mutagenesis system from Promega or QuikChange site-directed mutagenesis kit from Stratagene (Stratagene Europe, Amsterdam, The Netherlands) using the wild-type CXCR4 pcDNA3 Zeo expression vector as template according to the manufacturer's instructions. Each mutated clone (CXCR4.Y65F, CXCR4.Y76A, CXCR4.Y135F, and CXCR4.Y157A) was sequenced on an Applied Biosystems (Courtaboeuf, France) Model 373A automated sequencer, using Taq polymerase and dye terminator. CXCR4 and all the constructed mutants are tagged in the C-terminal part of the molecule with the FLAG epitope. A plasmid (pcDNA3 Zeo) encoding the HA tag epitope at the N terminus of wild-type CXCR4 (CXCR4.HA) was constructed by PCR and sequenced. Cell Culture and Transfection—HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen, Life Technologies), 1% penicillin-streptomycin, 1% glutamax. 107 cells were transfected with 5 μg of pcDNA Zeo containing the CXCR4 mutant genes (CXCR4.Y65F, CXCR4.Y76A, CXCR4.Y135F, and CXCR4.Y157A) using the FuGENE 6 transfection reagent (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. To construct stable HEK-293 cell lines expressing CXCR4 mutants containing deletions in ICL3, named CXCR4.ΔICL3-A, CXCR4.ΔICL3-B, and CXCR4.ΔICL3-C (55Brelot A. Heveker N. Montes M. Alizon M. J. Biol. Chem. 2000; 275: 23736-23744Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar), 107 cells were cotransfected with 5 μg of pcDNA containing the CXCR4 mutant genes and 0.5 μg of pcDNA Zeo. Stable clones able to grow in the presence of 250 μg/ml zeocin were selected by flow cytometry for receptor surface expression. Flow Cytometry—Cells (1 × 105) were incubated for 1 h at 4 °C with 50 μl of PBS or PBS supplemented with the appropriate mAb. After three washings with PBS, bound mAb was revealed by addition of 50 μl of a 1:100 dilution of fluorescein-conjugated (FITC) secondary immunoglobulin. After 1 h of staining, cells were washed with PBS, and fluorescence intensity at 543 nm was measured on a EPICS XL4-C cytofluorometer (Beckman-Coulter). To study the direct binding of SDF-1α to CXCR4 expressed on transfected HEK-293 cells, 106 cells were incubated in 30 μl of PBS for 1 h at 4 °C to prevent subsequent internalization, and 30 μl of a SDF-1α solution at 200 nm was then incubated for 20 min at 4 °C after cell centrifugation. After washing with PBS, 30 μl of an anti-SDF-1α antibody at 10 μg/ml was added for 30 min, and bound Ab was revealed as described previously (54Roland J. Murphy B.J. Ahr B. Robert-Hebmann V. Delauzun V. Nye K.E. Devaux C. Biard-Piechaczyk M. Blood. 2003; 101: 399-406Crossref PubMed Scopus (144) Google Scholar). Calcium Signaling—The technique used was described previously (54Roland J. Murphy B.J. Ahr B. Robert-Hebmann V. Delauzun V. Nye K.E. Devaux C. Biard-Piechaczyk M. Blood. 2003; 101: 399-406Crossref PubMed Scopus (144) Google Scholar). Briefly, 107 cells were resuspended in Hank's solution (Invitrogen, Life Technologies, Inc.), loaded with Fluo-3-AM at 2 μm for 20 min at room temperature and stimulated with buffer alone or SDF-1α at 250 nm. Ionomycin at 10–6m was then added to verify the capability of the cells to induce a calcium influx. Ratio of fluorescence of bound to free Fluo-3 was analyzed each 10 s on an EPICS XL4-C cytofluorometer. SDF-1α Activation and Western Blot Analysis—Cells were starved of serum during 2 days at 37 °C, 5% CO2. After washing three times in PBS, 5 × 106 cells/ml were resuspended in 100 μl of PBS and incubated at 37 °C for 15 min. Cells were then stimulated with SDF-1α at 125 nm for the specified times at 37 °C and lysed in 50 mm Tris (pH 8), 1% Triton X-100, 100 mm NaCl, 1 mm MgCl2, 2 mm benzamidine, 2 μg/ml leupeptin, 150 μm phenylmethylsulfonyl fluoride, containing NaF, Na3VO4 and β-glycerophosphate. The proteins were subjected to electrophoresis through 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Membranes were then blocked in Tris-buffered saline (TBS), 0.05% Tween 20, and 10% milk for 1 h at 20 °C. Blots were incubated overnight at 4 °C with the primary antibody diluted in TBS-Tween-5% milk. After 3 washings with TBS-Tween, the blots were incubated for 1 h at 20 °C with peroxidase-coupled antiserum diluted 1:2000 in TBS-Tween-5% milk. After further washing, the immune complexes were revealed by enhanced chemiluminescence (ECL, PerkinElmer Life Sciences) and subjected to autoradiography. Quantification of protein phosphorylation was performed by using the ImageJ program after autoradiography scanning. DNA Binding STAT3 Assay—After activation, cells were lysed in 50 mm Tris-HCl pH 7.9, 1% Nonidet P-40, 150 mm NaCl, 0.1 mm EDTA, 10 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 mg/ml aprotinin. The oligonucleotide sequence GTC-GACATTTCCCGTAAATC was used to immunopurify DNA binding STAT3. After incubation for 1 h at 4 °C of the cell lysate with 1 μg of double-stranded, 5′-biotinylated oligonucleotide coupled to 30 μl of a 50% suspension of streptavidin agarose. Complexes were washed twice in lysis buffer and affinity-purified proteins were fractionated by electrophoresis, transferred to PVDF membrane as described above. Western blot analysis was performed with anti-P-STAT3 Ab and immunoreactive bands were visualized by chemiluminescence. In Vitro Jak2 Kinase Assay—Cells were starved of serum over 1 day at 37 °C, 5% CO2, and transfected with 2 μg of pRK5-Jak2 by using the FuGENE 6 transfection reagent. The following day, cells were resuspended in 100 μl of PBS, stimulated with SDF-1α at 100 ng/ml for 1 min at 37 °C, and lysed in 50 mm Hepes (pH 7.5), 150 mm NaCl, 10 mm EDTA, 10 mm Na4P2O7, 100 mm NaF, 2 mm Na3VO4, 1 mm PMSF. After centrifugation, the pellet was resuspended in 500 μl of lysis buffer containing 1% Nonidet P-40 and incubated for 15 min at 4 °C. Jak2 was immunoprecipitated as described previously (54Roland J. Murphy B.J. Ahr B. Robert-Hebmann V. Delauzun V. Nye K.E. Devaux C. Biard-Piechaczyk M. Blood. 2003; 101: 399-406Crossref PubMed Scopus (144) Google Scholar). Briefly, 2 mg of proteins were used for immunoprecipitation with anti-Jak2 Ab and protein G-conjugated magnetic beads (Miltenyi Biotech) for 3 h at 4 °C. The immune complexes were then separated on magnetic columns and incubated in the presence of 2 μCi of [γ-32P]ATP in Tris buffer pH 7.4 containing 13.5 mm Mg2+ for 30 min at room temperature. After washing several times, phosphorylated products were eluted, loaded onto 10% SDS-PAGE, transferred onto PVDF membrane, and revealed by autoradiography. Phosphorylated Jak2 was identified by incubating the membrane with anti-Jak2 Ab. Membrane Protein Extraction—107 cells were washed in PBS and treated with 100 μg/ml of the membrane permeant cross-linking reagent DSP (3,3′-dithiopropionic acid di(N-hydroxysuccinimide ester)) for 1 h on ice. The reaction was stopped by adding PBS containing 100 mm glycine and 20 mm NEM (N-ethylmaleimide). After centrifugation for 5 min at 3000 × g, the pellet was resuspended in 150 μl of a lysis buffer containing 50 mm Tris-HCl pH 7.5, 500 mm NaCl, 5 mm EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitors, and incubated for 30 min at 4 °C. After two steps of freezing in liquid nitrogen/thawing at 37 °C and centrifugation for 30 min at 15,000 × g, the supernatant containing the membrane fraction was subjected to electrophoresis. Gels were transferred to PVDF membranes, and Western blot was performed as described previously. CXCR4 was revealed using the anti-FLAG Ab. Immunoprecipitation—107 cells stably expressing wild-type CXCR4 or CXCR4.ICL3m molecules containing a FLAG at the C terminus were transiently transfected with 5 μg of pcDNA Zeo containing CXCR4 tagged at the N terminus with the HA epitope (CXCR4.HA) by using the FuGENE 6 transfection reagent. After membrane protein extraction and lysate preclearing by incubation with protein A Sepharose, CXCR4.HA receptors were immunoprecipitated using an anti-HA Ab and protein A Sepharose. To analyze CXCR4 tyrosine phosphorylation after SDF-1α activation, phosphorylated proteins contained in cell lysates were immunoprecipitated using an anti-phosphotyrosine Ab, and analysis was performed as described above. Statistical Analysis—Variance analysis was performed after arc sine transformation of the data (56Zar J.H. Biostatistical Analysis. Prentice-Hall, Upper Saddle River, NJ1996Google Scholar). *, p < .05; **, p < .01; ***, p < .001. Construction and Expression of the CXCR4 Mutants—To investigate the role of intracellular tyrosines of CXCR4 in Jak2/STAT3 activation pathway after SDF-1α binding, CXCR4 mutants containing single amino acid substitutions in which Tyr65 and Tyr135 were replaced by a phenylalanine and Tyr76 and Tyr157 by alanine were constructed and stably transfected in HEK-293 cells. We also used previously constructed HEK-293 cells expressing CXCR4 mutants in which each entire intracellular loop was modified (CXCR4.ICL1m, CXCR4.ICL2m, and CXCR4.ICL3m) and a HEK-293 cell line expressing a truncated form of CXCR4 (CXCR4.7TM) (54Roland J. Murphy B.J. Ahr B. Robert-Hebmann V. Delauzun V. Nye K.E. Devaux C. Biard-Piechaczyk M. Blood. 2003; 101: 399-406Crossref PubMed Scopus (144) Google Scholar). Modification of ICL1 has suppressed Tyr65, but not Tyr76 and CXCR4.ICL2m has lost Tyr135 in the DRY box but Tyr157 is still present. All these CXCR4 mutants express a FLAG at the C terminus end of the receptor. Finally, to define the domains of ICL3 involved in Jak2/STAT3 activation, stably transfected HEK-293 cells expressing CXCR4 mutants containing deletions in ICL3, named CXCR4.ΔICL3-A, CXCR4.ΔICL3-B, and CXCR4.ΔICL3-C, described previously (55Brelot A. Heveker N. Montes M. Alizon M. J. Biol. Chem. 2000; 275: 23736-23744Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) were constructed. The amino acids 227–230, 230–233, and 233–236 are deleted in, respectively, CXCR4.ΔICL3-A, -B, and -C. A schematic representation of CXCR4 mutants used for this study is described in Fig. 1. This diagram is based on that proposed by Doranz et al. (60Doranz B.J. Orsini M.J. Turner J.D. Hoffman T.L. Berson J.F. Hoxie J.A. Peiper C. Brass L.F. Doms R.W. J. Virol. 1999; 73: 2752-2761Crossref PubMed Google Scholar). Expression of the CXCR4
DOI: 10.1186/1743-422x-9-69
2012
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Cited 20 times
HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4<sup>+</sup> T cell death
The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4+ T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4+ T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency.Abbreviations: Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA1: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.
2007
Cited 37 times
Mechanisms of CD4 T-cell depletion triggered by HIV-1 viral proteins.
Infection with HIV-1 leads to progressive CD4 T-cell death, resulting in AIDS development. The mechanisms that trigger this CD4 T-cell death are still not fully understood, but a lot of data indicates that apoptosis plays a major role in this cell demise. Both infected and uninfected CD4 T-cells can die during HIV-1 infection by different cell-death pathways, but HIV-1-induced, bystander, CD4 T-cell killing is now recognized as central to immunodeficiency. The HIV-1 directly modulates CD4 T-cell death using multiple different strategies in which several viral proteins have an essential role. Recent data demonstrate that relationships can exist between the three main types of programmed cell death, i.e. apoptosis, autophagic programmed cell death, and necrosis-like programmed cell death. Almost nothing is currently known about the role of necrosis-like programmed cell death in CD4 T-cell death induced by the viral proteins, but a very recent study demonstrates that autophagy is needed to trigger apoptosis of bystander CD4 T-cells, further increasing the level of complexity of this pathology. This review presents an overview of the major types of programmed cell death and details the mechanisms by which the HIV-1 viral proteins control both infected and uninfected CD4 T-cell death.
DOI: 10.1038/s41598-019-42075-3
2019
Cited 18 times
HIV-1 envelope glycoproteins isolated from Viremic Non-Progressor individuals are fully functional and cytopathic
Abstract In untreated HIV-1-infected individuals, viremia is positively associated with disease progression. However, some viremic non progressors (VNPs) individuals show paradoxical high CD4 + T cell counts. HIV-1 envelope glycoprotein complex (Env) is a major cytopathic determinant in viral replication; therefore, we have deeply characterized Env function in this rare clinical phenotype. Full-length Env clones isolated from individuals with Viral Load (VL) &gt; 10,000 copies/mL classified as VNPs (n = 15) or rapid progressors (RPs, n = 17) were geno- and phenotypically analyzed by determining diversity, expression, CD4 binding/signaling, fusogenicity, infectivity and autophagy induction. Selected Env clones from VNPs and RPs (n = 32) showed similar expression, fusion and infection abilities. Env clones from both groups showed similar affinity for CD4 during cell-to-cell transmission and consistently induced similar levels of CD4 signaling, measured by α-tubulin acetylation. Moreover, we demonstrate for the first time that primary Env clones from VNP and RP induce autophagy in uninfected cells and that this feature correlated with fusogenic capacity but was unrelated to disease progression. In conclusion, our data suggest that Env clones from VNP individuals are fully functional. Therefore, the paradoxical CD4 + T cell count stability coexisting with high levels of viral replication is unrelated to Env function.
DOI: 10.1002/pmic.200700306
2007
Cited 30 times
Proteomic analysis of the cellular responses induced in uninfected immune cells by cell-expressed X4 HIV-1 envelope
HIV-1 envelope gp120 and gp41 glycoproteins (Env), expressed at the cell surface, induce uninfected CD4 T-cell death, but the molecular mechanisms leading to this demise are still largely unknown. To better understand these events, we analyzed by a proteomic approach the differential protein expression profile of two types of uninfected immune cells after their coculture for 1-3 days with cells that express, or not, Env. First, umbilical cord blood mononuclear cells (UCBMCs) were used to approach the in vivo situation, i.e., blood uninfected naive cells that encounter infected cells. Second, we used the A2.01/CD4.403 T-cell line expressing wild type CXCR4 and a truncated form of CD4 that still undergoes Env-mediated apoptosis, independently of CD4 signaling. After coculture with cells expressing Env, 35 and 39 proteins presenting an altered expression in UCBMCs and the A2.01/CD4.403 T-cell line, respectively, were identified by mass-spectrometry. Whatever the cell type analyzed, the majority of these proteins are involved in degradation processes, redox homeostasis, metabolism and cytoskeleton dynamics, and linked to mitochondrial functions. This study provides new insights into the events that sequentially occur in bystander T lymphocytes after contact with HIV-infected cells and leading, finally, to apoptotic cell death.
DOI: 10.1089/hum.1997.8.10-1219
1997
Cited 35 times
<i>In Vitro</i>and<i>In Vivo</i>Secretion of Cloned Antibodies by Genetically Modified Myogenic Cells
In vivo production of recombinant antibodies by engineered cells may have applications for gene therapy of certain cancers and of certain severe viral diseases. It would also permit the development of new animal models of autoimmune diseases and new approaches for in vivo ablation of specific cell types for fundamental purposes. Using gene transfer of an anti-human thyroglobulin monoclonal antibody, we show here that several cell types permitting autologous grafting of genetically engineered cells are efficiently able to secrete antibodies in vitro. Those cells include skin fibroblasts, hepatocytes, and myogenic cells. We also show that the secreted antibodies display an affinity for the antigen close to that of the parental antibody, with, however, slight differences varying according to the cell type. This indicates that the foldings of antigen combining sites of antibodies produced in B cell- and non-B cell contexts are very similar. Finally, we report that, when implanted in the forelimb of a mouse, genetically modified myogenic cells are able to secrete antibodies for at least 4 months. Taken together, our observations point to the notion that genetic modification of patient cells may be used for long-term antibody-based gene therapies. Expression of cloned antibodies by genetically modified cells of patients may be used for developing surveillance treatments after primary anticancer therapies such as surgery, chemotherapies, or radiotherapies to avoid relapse or for inhibiting virus proliferation in the case of severe viral diseases. Because of their short life span and because of the fact that they already produce an antibody, plasmocytes from patients cannot be used for such a purpose. It is shown here that several cell types amenable to genetic modification and grafting to patients and which include skin fibroblasts, myogenic cells, and hepatocytes can produce cloned antibodies retaining the specificity and the affinity of the parental antibody. Grafting of engineered myogenic cells to mice also showed that delivery of recombinant antibodies in the blood stream was possible in vivo. This work casts the cellular basis for new antibody-based gene therapies and also more fundamental applications such as specific cell ablation and the study of certain aspects of autoimmunity.
DOI: 10.4161/auto.3275
2007
Cited 26 times
Autophagy and CD4<sup>+</sup>T lymphocyte destruction by HIV-1
The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection (i.e., the early stages following a new infection), whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.Addendum to:Autophagy is Involved in T Cell Death After Binding of HIV-1 Envelope Proteins to CXCR4L. Espert, M. Denizot, M. Grimaldi, V. Robert-Hebmann, B. Gay, M. Varbanov, P. Codogno and M. Biard-PiechaczykJ Clin Invest 2006; In press
DOI: 10.3389/fimmu.2012.00097
2012
Cited 19 times
Macroautophagy Regulation during HIV-1 Infection of CD4+ T Cells and Macrophages
Autophagy is an intracellular mechanism whereby pathogens, particularly viruses, are destroyed in autolysosomes after their entry into targets cells.Therefore, to survive and replicate in host cells, viruses have developed multiple strategies to either counteract or exploit this process.The aim of this review is to outline the known relationships between HIV-1 and autophagy in CD4+ T lymphocytes and macrophages, two main HIV-1 cell targets.The differential regulation of autophagy in these two cell-types is highlighted and its potential consequences in terms of viral replication and physiopathology discussed.
DOI: 10.1016/s0014-5793(98)00029-5
1998
Cited 28 times
Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties
A gene encoding a single‐chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 F ab , whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half‐lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.
DOI: 10.1016/0022-1759(96)00043-9
1996
Cited 25 times
Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis
We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and −κ chains combined with surface plasmon resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in koff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.
DOI: 10.4161/auto.5211
2008
Cited 16 times
What is the role of autophagy in HIV-1 infection?
HIV-1 infection is characterized by a progressive CD4 T cell depletion. It is now accepted that apoptosis of uninfected bystander CD4 T lymphocytes plays a major role in AIDS development. Viral envelope glycoproteins (Env) are mainly involved in inducing this cell death process, but the mechanisms triggered by HIV-1 leading to immunodeficiency are still poorly understood. Recently, we have demonstrated that autophagy is a prerequisite for Env-mediated apoptosis in uninfected CD4 T cells, underlining its role in HIV-1 infection. However, occurrence of autophagy in HIV-1-infected cells has not yet been described. Several hypotheses are discussed, based on the comparison with data from other viral infections.
DOI: 10.1111/boc.201100112
2012
Cited 12 times
HIV-1, ubiquitin and ubiquitin-like proteins: the dialectic interactions of a virus with a sophisticated network of post-translational modifications
Abstract The modification of intracellular proteins by ubiquitin (Ub) and ubiquitin‐like (UbL) proteins is a central mechanism for regulating and fine‐tuning all cellular processes. Indeed, these modifications are widely used to control the stability, activity and localisation of many key proteins and, therefore, they are instrumental in regulating cellular functions as diverse as protein degradation, cell signalling, vesicle trafficking and immune response. It is thus no surprise that pathogens in general, and viruses in particular, have developed multiple strategies to either counteract or exploit the complex mechanisms mediated by the Ub and UbL protein conjugation pathways. The aim of this review is to provide an overview on the intricate and conflicting relationships that intimately link HIV‐1 and these sophisticated systems of post‐translational modifications.
DOI: 10.1007/s10495-007-0106-4
2007
Cited 16 times
The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells
DOI: 10.1007/978-3-642-00302-8_15
2009
Cited 14 times
Autophagy in HIV-Induced T Cell Death
HIV infection leads to progressive CD4 T cell depletion, resulting in the development of AIDS. The mechanisms that trigger T cell death after HIV infection are still not fully understood, but a lot of data indicate that apoptosis of uninfected CD4 lymphocytes plays a major role. HIV directly modulates cell death using various strategies in which several viral proteins, in particular the envelope glycoproteins (Env), play an essential role. Importantly, Env, expressed on infected cells, triggers autophagy in uninfected CD4 T cells, leading to their apoptosis. Furthermore, HIV, like other viruses, has evolved strategies to inhibit this autophagic process in HIV-infected cells. This discovery further increases the level of complexity of the cellular processes involved in HIV-induced pathology. Interestingly, HIV protease inhibitors, currently used in highly active antiretroviral therapy (HAART), are able to induce autophagy in cancer cells, leading to a recent repositioning of these drugs as anticancer agents. This review presents an overview of the relationship between HIV, HAART, and autophagy.
DOI: 10.1111/boc.201900071
2019
Cited 10 times
HIV‐1 Vpr inhibits autophagy during the early steps of infection of CD4 T cells
Background information Autophagy is induced during HIV‐1 entry into CD4 T cells by the fusion of the membranes triggered by the gp41 envelope glycoprotein. This anti‐HIV‐1 mechanism is inhibited by the viral infectivity factor (Vif) neosynthesized after HIV‐1 integration to allow viral replication. However, autophagy is very rapidly controlled after HIV‐1 entry by a still unknown mechanism. As HIV‐1 viral protein R (Vpr) is the only auxiliary protein found within the virion in substantial amount, we studied its capability to control the early steps of HIV‐1 envelope‐mediated autophagy. Results We demonstrated that ectopic Vpr inhibits autophagy in both the Jurkat CD4 T cell line and HEK.293T cells. Interestingly, Vpr coming from the virus also blocks autophagy in CD4 T cells, the main cell target of HIV‐1. Furthermore, Vpr decreases the expression level of two essential autophagy proteins (ATG), LC3B and Beclin‐1, and an important autophagy‐related protein, BNIP3 as well as the level of their mRNA. We also demonstrated in HEK.293T cells that Vpr degrades the FOXO3a transcription factor through the ubiquitin proteasome system. Conclusion Vpr, the only well‐expressed HIV‐1 auxiliary protein incorporated into viruses, is able to negatively control autophagy induced during HIV‐1 entry into CD4 T cells. Significance We provide insights of how HIV‐1 controls autophagy very early after its entry into CD4 T cells and discovered a new function of Vpr. These results open the route to a better understanding of the roles of Vpr during HIV‐1 infection through FOXO3a degradation and could be important to consider additional therapies that counteract the role of Vpr on autophagy.
DOI: 10.1016/s0165-2478(99)00124-8
1999
Cited 18 times
Role of CXCR4 in HIV-1-induced apoptosis of cells with a CD4+, CXCR4+ phenotype
DOI: 10.1051/medsci/20062289677
2006
Cited 12 times
Autophagie et destruction des lymphocytes T CD4 par le VIH-1
DOI: 10.1097/qai.0000000000001201
2017
Cited 7 times
Brief Report
Abstract: Autophagy restricts infection of CD4 T lymphocytes by HIV-1, but little is known about autophagy in treated HIV-1–infected individuals. We have analyzed the capability of CD4 T cells from aviremic-treated individuals to trigger autophagy and correlated this response with parameters known to be important for immunological recovery. Autophagy was significantly decreased in CD4 T cells from HIV-1–treated individuals compared with uninfected controls, and this defective autophagic response was more pronounced in individuals with poor CD4 T-cell recovery, suggesting a link between impaired autophagy in CD4 T cells and chronic immunological defects that remain in treated HIV infection.
DOI: 10.1051/medsci/20173303019
2017
Cited 7 times
Autophagie, protéines ATG et maladies infectieuses
One of the main functions of the autophagy pathway is to control infections. Intracellular micro-organisms or their products once internalized in the host cell can be directly degraded by autophagy, a process called xenophagy. Autophagy is also involved in other innate immune responses and participates to the adaptive immune system. In addition, several autophagy proteins play a role in the development of infectious diseases independently of their role in the autophagy pathway. To replicate efficiently, pathogens have therefore evolved to counteract this process or to exploit it to their own profit. The review focuses on the relationship between autophagy and micro-organisms, which is highly diverse and complex. Many research groups are now working on this topic to find new therapeutics and/or vaccines. Given the large number of data, we have addressed this subject through some representative examples.
DOI: 10.1051/medsci/2019129
2019
Cited 6 times
La phagocytose associée à LC3 (LAP)
Phagocytosis and macroautophagy, named here autophagy, are two essential mechanisms of lysosomal degradation of diverse cargos into membrane structures. Both mechanisms are involved in immune regulation and cell survival. However, phagocytosis triggers degradation of extracellular material whereas autophagy engulfs only cytoplasmic elements. Furthermore, activation and maturation of these two processes are different. LAP (LC3-associated phagocytosis) is a form of phagocytosis that uses components of the autophagy pathway. It can eliminate (i) pathogens, (ii) immune complexes, (iii) threatening neighbouring cells, dead or alive, and (iv) cell debris, such as POS (photoreceptor outer segment) and the midbody released at the end of mitosis. Cells have thus optimized their means of elimination of dangerous components by sharing some fundamental elements coming from the two main lysosomal degradation pathways.La phagocytose associée à LC3 (LAP) - Phagocytose ou autophagie ?Phagocytose et macroautophagie, appelée ici autophagie, sont deux mécanismes essentiels de dégradation lysosomale de divers cargos englobés dans des structures membranaires. Ils sont tous deux impliqués dans la régulation du système immunitaire et la survie cellulaire. Cependant, la phagocytose permet l’ingestion de matériel extracellulaire alors que l’autophagie dégrade des composants intra-cytoplasmiques, avec des mécanismes d’activation et de maturation différents. La LAP (LC3-associated phagocytosis) est une forme particulière de phagocytose qui utilise certains éléments de l’autophagie. Elle permet l’élimination de pathogènes, de complexes immuns, de cellules avoisinantes, mortes ou vivantes, constituant un danger pour l’organisme, et de débris cellulaires, tels que les segments externes des photorécepteurs (POS, photoreceptor outer segment), ou la pièce centrale du pont intercellulaire produit en fin de mitose. Les cellules ont ainsi « optimisé » leurs moyens d’éliminer les composés potentiellement dangereux en partageant certains éléments essentiels des deux voies de dégradation lysosomale.
DOI: 10.1016/1380-2933(95)00004-6
1995
Cited 14 times
Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay
A high affinity anti-digoxin single-chain Fv antibody fragment (scFv) was cloned from the mouse 2C2 hybridoma cell line and was functionally expressed both in the Escherichia coli periplasm as a soluble molecule and at the surface of the filamentous M13 bacteriophage as a fusion protein with the gene III minor coat protein. The 2C2 scFv sequence significantly differs from that of all the other anti-digoxin antibodies previously described. The 2C2 scFv shares with its parental monoclonal antibody a high specificity for digoxin, a cross-reactivity with active digoxin metabolites, but none with inactive metabolites. M13 phages displaying the 2C2 scFv at their surface have a high apparent affinity constant for digoxin (6.6 x 10(8) M-1) and were directly used to set up a novel type of immunoenzymatic assay for monitoring digoxin in sera of patients treated for either congestive heart failure or cardiac arrythmias. We thus report for the first time that phages displaying scFv may constitute a large source of important new reagents in the field of immunodiagnosis.
DOI: 10.2144/97233rr03
1997
Cited 12 times
In-Cell Assembly of scFv from Human Thyroid- Infiltrating B Cells
The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, to our knowledge, for the first time to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19 + B cells isolated from human thyroid tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the CreloxP system, we obtained a unique 800-bp band corresponding in size to scFv fragments. We provide evidence that recombination between VH and VL genes occurred inside the same cell. This in-cell amplification and association procedure is a potentially useful tool for the study of autoantibody gene families and the VH/VL pairing that occurs during the autoimmune process.
2005
Cited 6 times
Mitochondria-dependent apoptosis in T-cell homeostasis.
T-cell homeostasis is maintained by balancing the proliferation and destruction of lymphocytes at multiple steps during the life of an individual. Regulated mitochondria-dependent apoptosis is essential for both the development and the subsequent maintenance of the immune system, in that it keeps the total number of lymphocytes constant. Firstly, during thymic development, sequential stages of T-cell maturation require strict control of T-cell selection, and secondly, apoptosis is essential in controlling the massive expansion of antigen-specific T-cells after their activation. Failure in each of these steps can lead to pathologies, while drugs that target apoptosis could have therapeutic benefit.
DOI: 10.1089/hyb.1995.14.443
1995
Cited 7 times
Cloning and Expression of a Single-Chain Antibody Fragment Specific for a Monomorphic Determinant of Class I Molecules of the Human Major Histocompatibility Complex
B9.12.1 is a monoclonal antibody specific for a monomorphic determinant of human MHC class I molecules. It is currently used for cell typing and is useful for targeting infection of human cells by murine ecotropic retroviruses. We have cloned and expressed it in the form of a single-chain variable fragment (ScFv) that recognizes the same epitope as the parental antibody. Through genetic engineering, this ScFv may be used for developing new cell-typing probes and new retroviral targeting approaches.
2019
[LAP (LC3-associated phagocytosis): phagocytosis or autophagy?]
Phagocytosis and macroautophagy, named here autophagy, are two essential mechanisms of lysosomal degradation of diverse cargos into membrane structures. Both mechanisms are involved in immune regulation and cell survival. However, phagocytosis triggers degradation of extracellular material whereas autophagy engulfs only cytoplasmic elements. Furthermore, activation and maturation of these two processes are different. LAP (LC3-associated phagocytosis) is a form of phagocytosis that uses components of the autophagy pathway. It can eliminate (i) pathogens, (ii) immune complexes, (iii) threatening neighbouring cells, dead or alive, and (iv) cell debris, such as POS (photoreceptor outer segment) and the midbody released at the end of mitosis. Cells have thus optimized their means of elimination of dangerous components by sharing some fundamental elements coming from the two main lysosomal degradation pathways.
DOI: 10.7202/013766ar
2006
Autophagie et destruction des lymphocytes T CD4 par le VIH-1
> La mort cellulaire programmee (PCD, programmed cell death) est un processus physiologique essentiel pour le maintien de l’homeostasie. Au cours de l’evolution, plusieurs mecanismes de mort, souvent intriques, se sont mis en place pour assurer au mieux la survie des organismes [1]. L’absence de PCD peut contribuer au developpement de pathologies telles que le cancer et, de facon inverse, une PCD excessive peut aussi devenir pathologique, comme c’est le cas pour l’immunodeficience provoquee par le VIH-1 (virus de l’immunodeficience humaine). Cela montre bien la necessite d’un tres haut niveau de regulation entre survie et mort cellulaire. Deux types majeurs de PCD sont connus : la PCD de type I ou apoptose et la PCD de type II, ou mort autophagique [2, 3]. L’autophagie (Figure 1) est un mecanisme physiologique hautement regule dependant des genes ATG (autophagy-related genes), faisant intervenir deux systemes de conjugaison semblables au processus d’ubiquitination (formation des conjugues Atg12-Atg5-Atg16 et Atg8/LC3-PE), et de plusieurs voies de signalisation [4]. La decouverte des genes ATG chez la levure a permis de reconsiderer ce processus longtemps laisse de cote par manque d’outils [5]. L’autophagie a pour fonction primaire de reguler le recyclage des proteines a duree de vie longue et des organelles cellulaires. Ce processus est caracterise par une degradation de composes cytoplasmiques a l’interieur de vacuoles appelees autophagosomes. Les autophagosomes fusionnent avec les lysosomes pour former des autophagolysosomes qui sont les compartiments terminaux de la degradation [4]. La phosphatidylinositol 3 -kinase de classe III est impliquee dans les phases precoces de la formation des vacuoles et controle la voie autophagique en s’associant avec la proteine ATG6/ Beclin 1 au niveau du reseau trans-golgien [6]. Les mecanismes moleculaires impliques dans la formation des autophagosomes et regules par ce complexe de signalisation sont encore indetermines. L’autophagie est un processus complexe car il peut conduire soit a la survie, soit a la mort cellulaire [7]. De facon interessante, de plus en plus de travaux montrent que l’autophagie et l’apoptose sont des processus etroitement lies. En effet, l’autophagie peut etre independante de l’apoptose, l’inhiber ou Autophagie et destruction des lymphocytes T CD4 par le VIH-1
DOI: 10.3233/hab-1999-9308
1999
Cited 4 times
Analysis of vH and vL genes of a monospecific human anti-myosin antibody produced by a B cell from the primary repertoire
Epstein-Barr virus (EBV) transformation of B lymphocytes from a Glanzmann's thrombasthenia patient with a serum antibody to the integrin alpha IIb beta 3, led to the immortalization of a B cell secreting a monospecific IgM monochonal antibody (MAb), B7, reactive with platelet myosin. Analysis of B7 V genes revealed minimally mutated sequences: the immortalized B cell issued from the primary repertoire, with no evidence of an in vivo selection by myosin. The V genes were here compared with sequences of human MAbs available on databases to more clearly understand the monospecificity of the B7 MAb. B7 V genes were closely identical to rearranged V genes in clones with self-specificities, often secreting polyreactive antibodies. In contrast, B7 is an unmutated monoreactive human MAb able to recognize myosin with a high avidity. Comparison of the CDR3H sequence with that of MAbs in databases supports a central role for the CDR3H subdomain in determining monospecificity. Our results suggest the existence of a monospecific autoreactive B cell compartment, besides the well-known polyspecific one, susceptible to be the template of pathogenic autoreactivity, characterized by antibodies of high affinity and specificity.
DOI: 10.1101/2019.12.19.881110
2019
HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4+ T cell death
Abstract The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4+ T lymphocytes death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers autophagy, process necessary to subsequent apoptosis, and to production of Reactive Oxygen Species (ROS) in bystander CD4+ T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, catalase and PEX14, upon Env exposure, since the down-regulation of either BECLIN 1 or p62/SQSTM1 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency.
DOI: 10.1016/s0014-5793(99)01308-3
1999
Analysis of the V genes coding for a monospecific human antibody to myosin and functional expression of single chain Fv fragments
A monospecific human IgM monoclonal antibody (mAb), reactive with myosin from human heart, has been obtained by EBV transformation. This mAb may have a diagnostic potential in the imaging of myocardial necrosis. However, owing to the fact that the molecular mass of an IgM is 900 kDa, a poor diffusion and a slow penetration inside necrotic myocytes could reduce its capacity for scintigraphic detection. In order to alleviate these problems, we constructed the scFv by cloning the VH and VL domains into the pHOG21 vector. Analysis of the V genes proved an unmutated configuration showing that the immortalized B cell issued from the primary IgM repertoire. The expression product in Escherichia coli was a 35 kDa scFv fragment with the antigen-binding specificity of the parental mAb.
DOI: 10.3233/hab-1999-9108
1999
Human single-chain Fv fragments from a combinatorial library using the loxP-Cre recombination system
A human scFv display library has been constructed from peripheral blood lymphocytes of a patient suffering from Hashimoto's thyroiditis. Upon induction of Cre recombinase, the amplified VH and VL genes were recombined via two loxP sites inserted in amplification primers to construct in vitro scFv genes. Either soluble scFvs or scFvs displayed on phage were screened for binding to human thyroglobulin after two pannings with this antigen. Three scFvs were obtained which showed very similar nucleotidic sequences. The VH genes expressed display 96.4% nucleotide sequence homology with the germline VH251 gene, one of the two functional members of the small VH5 family and are mutated in sites already described as "selectively neutral" mutations and the VL genes are close to the germline DPL8 gene. These scFvs bind not only to human thyroglobulin but also to other self and exogenous antigens.
DOI: 10.1016/b978-0-12-405877-4.00007-x
2014
Induction of Autophagy in HIV-1-Uninfected Cells
Abstract Autophagy is recognized as an innate mechanism by which intracellular pathogens are degraded into autolysosomes. In consequence, to avoid their destruction, many pathogens have evolved to block at least one step of the autophagy process or to exploit autophagic membranes for their own self-serving purposes. Autophagy is triggered by molecules that sense a danger, such as pathogen-recognition receptors (PRRs), damage-associated molecular pattern molecules (DAMPs), pathogen receptors, and cytokines. Interestingly, several enveloped viruses can induce autophagy through membrane-fusion events that occur at the entry step of their life cycle. For human immunodeficiency virus type 1 (HIV-1), the envelope glycoprotein gp41 is responsible for the fusion between the membrane of the virus, or the infected cell, and the membrane of the uninfected target cell. This fusion event triggers autophagy in CD4 T lymphocytes, leading to their apoptosis – the mechanism responsible for development of AIDS. Thus, autophagy plays an important role in the pathogenesis of HIV-1 infection by killing specifically the uninfected CD4 T lymphocytes through membrane-fusion events.
DOI: 10.1007/978-1-4614-9610-6_395-1
2015
Autophagy and HIV Infection
DOI: 10.1016/b978-0-12-405877-4.00032-9
2014
Contributors
DOI: 10.1016/j.meegid.2011.07.001
2011
Montpellier Infectious Diseases (MID): 1st Annual Meeting (2011)
DOI: 10.4161/auto.8.4.19496
2012
Guidelines for the use and interpretation of assays for monitoring autophagy.
DOI: 10.1186/1742-4690-6-s2-p36
2009
Autophagy plays an essential role in HIV-1 infection
HIV-1 can infect and replicate in both CD4 T cells and macrophages, and direct cell-to-cell spread is an important route of HIV-1 propagation. It requires interaction between HIV-1 envelope glycoproteins (Env, composed of gp120 and gp41), expressed at the surface of infected cells, and HIV-1 receptors, CD4 and a coreceptor, on the target cells. The gp120 interacts first with CD4, which triggers conformational changes leading to increased exposure of gp120 regions (including the V3 loop) able to bind to the coreceptor, mainly CCR5 or CXCR4. This interaction induces a structural rearrangement in gp41, insertion of its N terminus fusion peptide into the target membrane, and fusion. R5 and X4 HIV-1 strains use CCR5 and CXCR4, respectively, for entry. We have previously demonstrated that, independently of HIV-1 replication, X4 HIV-infected cells trigger autophagy in the uninfected CD4 T lymphocytes. Env-mediated autophagy is dependent on the gp41 fusogenic activity but is independent of a direct CD4- and CXCR4-mediated signaling pathway. Furthermore, this autophagy process is required to trigger CD4 T cell apoptosis since blockade of autophagy at different steps, by either drugs or short interfering RNAs specific for autophagy genes, totally inhibits Env-mediated apoptosis. Our last results show that autophagy and cell death are also induced in the uninfected CD4 T cells by HIV-1 R5 Env, while autophagy is inhibited in productively X4 or R5-infected CD4 T cells. In contrast, uninfected macrophages, a preserved cell population during HIV-1 infection, do not undergo X4 or R5 Env-mediated autophagy. Autophagosomes, however, are present in macrophages exposed to infectious HIV-1 particles, independently of coreceptor use. Interestingly, two populations of autophagic macrophages can be observed during their coculture with HIV-1-infected cells: one highly autophagic and the other weakly autophagic. Surprisingly, viruses could be detected in the weakly autophagic cells but not in the highly autophagic cells. In addition, we show that the triggering of autophagy in macrophages is necessary for viral replication but addition of Bafilomycin A1, which blocks the final stages of autophagy, strongly increases productive infection. Taken together, our data suggest that autophagy plays a complex, but essential, role in HIV pathology by regulating both viral replication and the fate of the target cells.
DOI: 10.6084/m9.figshare.13109835
2022
HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4<sup>+</sup> T cell death
The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4<sup>+</sup> T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4<sup>+</sup> T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency. <b>Abbreviations:</b> Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA<sub>1</sub>: bafilomycin A<sub>1</sub>; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.
DOI: 10.1007/978-1-4939-7101-5_395
2018
Autophagy and HIV Infection
DOI: 10.6084/m9.figshare.13109835.v1
2020
HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4<sup>+</sup> T cell death
The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4<sup>+</sup> T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4<sup>+</sup> T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency. <b>Abbreviations:</b> Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA<sub>1</sub>: bafilomycin A<sub>1</sub>; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.
DOI: 10.6084/m9.figshare.13109835.v2
2021
HIV-1 Env induces pexophagy and an oxidative stress leading to uninfected CD4<sup>+</sup> T cell death
The immunodeficiency observed in HIV-1-infected patients is mainly due to uninfected bystander CD4<sup>+</sup> T lymphocyte cell death. The viral envelope glycoproteins (Env), expressed at the surface of infected cells, play a key role in this process. Env triggers macroautophagy/autophagy, a process necessary for subsequent apoptosis, and the production of reactive oxygen species (ROS) in bystander CD4<sup>+</sup> T cells. Here, we demonstrate that Env-induced oxidative stress is responsible for their death by apoptosis. Moreover, we report that peroxisomes, organelles involved in the control of oxidative stress, are targeted by Env-mediated autophagy. Indeed, we observe a selective autophagy-dependent decrease in the expression of peroxisomal proteins, CAT and PEX14, upon Env exposure; the downregulation of either BECN1 or SQSTM1/p62 restores their expression levels. Fluorescence studies allowed us to conclude that Env-mediated autophagy degrades these entire organelles and specifically the mature ones. Together, our results on Env-induced pexophagy provide new clues on HIV-1-induced immunodeficiency. <b>Abbreviations:</b> Ab: antibodies; AF: auranofin; AP: anti-proteases; ART: antiretroviral therapy; BafA<sub>1</sub>: bafilomycin A<sub>1</sub>; BECN1: beclin 1; CAT: catalase; CD4: CD4 molecule; CXCR4: C-X-C motif chemokine receptor 4; DHR123: dihydrorhodamine 123; Env: HIV-1 envelope glycoproteins; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFP-SKL: GFP-serine-lysine-leucine; HEK: human embryonic kidney; HIV-1: type 1 human immunodeficiency virus; HTRF: homogeneous time resolved fluorescence; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NAC: N-acetyl-cysteine; PARP: poly(ADP-ribose) polymerase; PEX: peroxin; ROS: reactive oxygen species; siRNA: small interfering ribonucleic acid; SQSTM1/p62: sequestosome 1.
2001
Binding of human immunodeficiency virus type 1 gp120 to CXCR4 induces mitochondrial transmembrane depolarization and cytochrome c-mediated apoptosis independently of Fas signaling
DOI: 10.1002/(sici)1099-1352(199812)11:1/6<117::aid-jmr404>3.0.co;2-o
1998
Affinity of recombinant antibody and antibody fragment binding to human thyroglobulin: potential applications in gene therapy
Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant antihuman thyroglobulin (hTg) antibody.
DOI: 10.1016/0923-2532(96)80503-5
1995
Introduction à l'ingénierie génétique des anticorps
L'ingénierie moléculaire des sites anticorps connaît un essor considérable depuis le développement des systèmes d'expression dans Escherichia coli. Il est maintenant possible de produire des fragments d'anticorps à partir d'hybridomes ou de populations hétérogènes de lymphocytes B. Cette nouvelle technologie va permettre l'exploration immunologique de répertoires (naïfs ou stimulés par l'antigène) de gènes codant pour les domaines variables des anticorps humains ou animaux. Antibody engineering has received a boost from the development of an Escherichia coli expression system that now allows the production of fragment antibodies from either an hybridoma or an heterogeneous population of B lymphocytes. The development of expression libraries opens the door to the exploration of diverse repertoires of V genes derived from immunized or non-immunized humans or animals.