Biomedical subjects

Guillaume Bossis

Publications and source records attributed to Guillaume Bossis.

14 recordsLinked to original sources

PubMed2006

E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation.

p53 is regulated by multiple posttranslational modifications, including Hdm2-mediated ubiquitylation that drives its proteasomal degradation. Here, we identify the p53-associated factor E4F1, a ubiquitously expressed zinc-finger protein first identified as a cellular target of the viral oncoprotein E1A, as an atypical ubiquitin E3 ligase for p53 that modulates its effector functions without promoting proteolysis. E4F1 stimulates oligo-ubiquitylation in the hinge region of p53 on lysine residues distinct from those targeted by Hdm2 and previously described to be acetylated by the acetyltransferase PCAF. E4F1 and PCAF mediate mutually exclusive posttranslational modifications of p53. E4F1-dependent Ub-p53 conjugates are associated with chromatin, and their stimulation coincides with the induction of a p53-dependent transcriptional program specifically involved in cell cycle arrest, and not apoptosis. Collectively, our data reveal that E4F1 is a key posttranslational regulator of p53, which modulates its effector functions involved in alternative cell fates: growth arrest or apoptosis.

Acetylation↗

PubMed2006

SUMO: regulating the regulator.

Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier) family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i) the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii) global regulation of SUMO conjugation and deconjugation.

Journal Article↗

PubMed2006

Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes.

Posttranslational modification with small ubiquitin-related modifier (SUMO) has emerged as a central regulatory mechanism of protein function. However, little is known about the regulation of sumoylation itself. It has been reported that it is increased after exposure to various stresses including strong oxidative stress. Conversely, we report that ROS (reactive oxygen species), at low concentrations, result in the rapid disappearance of most SUMO conjugates, including those of key transcription factors. This is due to direct and reversible inhibition of SUMO conjugating enzymes through the formation of (a) disulfide bond(s) involving the catalytic cysteines of the SUMO E1 subunit Uba2 and the E2-conjugating enzyme Ubc9. The same phenomenon is also observed in a physiological scenario of endogenous ROS production, the respiratory burst in macrophages. Thus, our findings add SUMO conjugating enzymes to the small list of specific direct effectors of H(2)O(2) and implicate ROS as key regulators of the sumoylation-desumoylation equilibrium.

Animals↗

PubMed2005

Mechanisms of delivery of ubiquitylated proteins to the proteasome: new target for anti-cancer therapy?

The proteasome is the main proteolytic machinery of the cell. It is responsible for the basal turnover of many intracellular polypeptides, the elimination of abnormal proteins and the generation of the vast majority of peptides presented by class I major histocompatibility complex molecules. Proteasomal proteolysis is also involved in the control of virtually all cellular functions and major decisions through the spatially and timely regulated destruction of essential cell regulators. Therefore, the elucidation of its molecular mechanisms is crucial for the full understanding of the physiology of cells and whole organisms. Conversely, it is increasingly clear that proteasomal degradation is either altered in numerous pathological situations, including many cancers and diseases resulting from aberrant cell differentiation, or instrumental for the development of these pathologies. This, consequently, makes it an attractive target for therapeutical intervention. There is ample evidence that most cell proteins must be polyubiquitylated prior to proteasomal degradation. If the structure and the mode of functioning of the proteasome, as well as the enzymology of ubiquitylation, are relatively well understood, how substrates are delivered to and recognized by the proteolytic machine has remained mysterious till recently. The recent literature indicates that the mechanisms involved are multiple, complex and exquisitely regulated and provides new potential targets for anti-cancer pharmacological intervention.

Animals↗

PubMed2005

A fluorescence resonance energy transfer-based assay to study SUMO modification in solution.

Analysis of posttranslational modifications with ubiquitin and ubiquitin-related proteins (Ubl) generally involves detection of the modified species by immunoblotting or autoradiography, techniques that are not easily applicable for kinetic, quantitative, or high-throughput assays. To circumvent these limitations for studies on ubiquitin-related proteins of the SUMO family, we have developed a fluorescence resonance energy transfer (FRET)-based assay system using yellow fluorescent protein (YFP)-tagged mature SUMO1 (amino acids 1-97) and cyan fluorescent protein (CFP)-tagged RanGAP1 (amino acids 400-589) as model substrates. Reactions are set up in 384-well microtiter plates and are followed online using a fluorescence microtiter plate reader. Applications may involve identification and analysis of SUMO-modifying enzymes and isopeptidases, comparison of enzyme and substrate mutants, and screens for small molecular weight inhibitors. The principal outline of the assay should be applicable to other Ubl conjugation systems as well.

Animals↗

PubMed2005

[Proteasomal degradation: from addressing of substrates to therapeutical perspectives].

The proteasome is the main intracellular proteolytic machinery. It is involved in all major cellular functions and decisions. It has long been thought that prior ubiquitinylation of almost all of its substrates was necessary for degradation. It has also long been considered that ubiquitinylation and degradation were two uncoupled mechanisms and that the recruitment of ubiquitinylated species was only performed by specialized subunits of the proteasome. The recent literature questions this simplified view. It also suggests that, on the one hand, the fraction of proteins hydrolyzed by the proteasome independently of their ubiquitinylation has largely been underestimated and, on the other hand, that the recognition of ubiquitinylated proteins involves complex addressing systems. Furthermore, it indicates a higher order structuration of the ubiquitin/proteasome pathway, a fraction of the proteasome and of ubiquitinylation enzymes being engaged in supramolecular complexes. Finally, proteasomal degradation is altered in a number of pathological situations. It, thus, constitutes a therapeutic target and the first applications are emerging.

Animals↗

PubMed2005

Down-regulation of c-Fos/c-Jun AP-1 dimer activity by sumoylation.

The inducible transcriptional complex AP-1, composed of c-Fos and c-Jun proteins, is crucial for cell adaptation to many environmental changes. While its mechanisms of activation have been extensively studied, how its activity is restrained is poorly understood. We report here that lysine 265 of c-Fos is conjugated by the peptidic posttranslational modifiers SUMO-1, SUMO-2, and SUMO-3 and that c-Jun can be sumoylated on lysine 257 as well as on the previously described lysine 229. Sumoylation of c-Fos preferentially occurs in the context of c-Jun/c-Fos heterodimers. Using nonsumoylatable mutants of c-Fos and c-Jun as well as a chimeric protein mimicking sumoylated c-Fos, we show that sumoylation entails lower AP-1 transactivation activity. Interestingly, single sumoylation at any of the three acceptor sites of the c-Fos/c-Jun dimer is sufficient to substantially reduce transcription activation. The lower activity of sumoylated c-Fos is not due to inhibition of protein entry into the nucleus, accelerated turnover, and intrinsic inability to dimerize or to bind to DNA. Instead, cell fractionation experiments suggest that decreased transcriptional activity of sumoylated c-Fos is associated with specific intranuclear distribution. Interestingly, the phosphorylation of threonine 232 observed upon expression of oncogenically activated Ha-Ras is known to superactivate c-Fos transcriptional activity. We show here that it also inhibits c-Fos sumoylation, revealing a functional antagonism between two posttranslational modifications, each occurring within a different moiety of a bipartite transactivation domain of c-Fos. Finally we report that the sumoylation of c-Fos is a dynamic process that can be reversed via multiple mechanisms. This supports the idea that this modification does not constitute a final inactivation step that necessarily precedes protein degradation.

Binding Sites↗

PubMed2004

SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1.

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling.

Animals↗

PubMed2003

The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression.

c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and basic domain/leucine zipper-dependent mechanisms. Taken together, our data show that multiple degradation mechanisms, differing according to the conditions of expression, may operate on c-Fos to ensure a proper level and/or timing of expression. Moreover, they also indicate that the half-life of c-Fos during the G0/G1 phase transition is determined by a delicate balance between opposing stabilizing and destabilizing mechanisms operating at the same time.

Animals↗

PubMed2003

c-Fos proto-oncoprotein is degraded by the proteasome independently of its own ubiquitinylation in vivo.

Prior ubiquitinylation of the unstable c-Fos proto-oncoprotein is thought to be required for recognition and degradation by the proteasome. Contradicting this view, we report that, although c-Fos can form conjugates with ubiquitin in vivo, nonubiquitinylatable c-Fos mutants show regulated degradation identical to that of the wild-type protein in living cells under two classical conditions of study: transient c-fos gene expression during the G(0)/G(1) phase transition upon stimulation by mitogens and constitutive expression during asynchronous growth. Moreover, c-Fos destruction during the G(0)/G(1) phase transition is unusual because it depends on two distinct but cumulative mechanisms. We report here that one mechanism involves a C-terminal destabilizer which does not need an active ubiquitin cycle, whereas the other involves an N-terminal destabilizer dependent on ubiquitinylation of an upstream c-Fos breakdown effector. In addition to providing new insights into the mechanisms of c-Fos protein destruction, an important consequence of our work is that ubiquitinylation-dependent proteasomal degradation claimed for a number of proteins should be reassessed on a new experimental basis.

Cell Division↗

PubMed2002

CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion.

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.

Bacterial Toxins↗

PubMed2002

NF-kappaB activation upon interaction of HIV-1 envelope glycoproteins with cell surface CD4 involves IkappaB kinases.

Human immunodeficiency virus type-1 envelope glycoprotein (gp120(env)) binding to cell surface CD4 receptor triggers a broad range of intracellular effects leading to T cell activation and cell cycle entry. Among these effects we and others previously reported on the nuclear translocation of the nuclear factor-kappaB (NF-kappaB) transcription factor. The present work further investigates the signal transduction pathways involved in gp120(env)-induced NF-kappaB activation. We demonstrate that gp120(env)-CD4 interaction stimulates the hyperphosphorylation of IkappaB-alpha inhibitory protein. Conversely, overexpression of a dominant-negative IkappaB-alpha transgene mutated at S32 and S36 residues, abolishes gp120(env)-induced NF-kappaB activation. IkappaB kinases (IKKs) activity was found to be selectively enhanced following CD4 engagement with gp120(env) and to mediate the phosphorylation of IkappaB-alpha while co-transfection experiments using dominant-negative forms of IKKs inhibited gp120(env)-induced NF-kappaB activation. Taken together, these results confirm that IKKs complex play a key role in gp120(env)-induced NF-kappaB activation.

CD4 Antigens↗

PubMed2002

Evasion from proteasomal degradation by mutated Fos proteins expressed from FBJ-MSV and FBR-MSV osteosarcomatogenic retroviruses.

c-Fos proto-oncoprotein is highly unstable, which is crucial for rapid gene expression shut-off and control of its intrinsic oncogenic potential. It is massively degraded by the proteasome in vivo in various situations. Although there is evidence that c-Fos can be ubiquitinylated in vitro, the unambiguous demonstration that ubiquitinylation is necessary for recognition and subsequent hydrolysis by the proteasome in vivo is still lacking. Moreover, genetic analysis have also indicated that c-Fos can be addressed to the proteasome via different mechanisms depending on the conditions studied. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses under mutated forms which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of a C-terminal c-Fos destabilizer but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. Though mutations in viral Fos proteins confer full resistance to proteasomal degradation, stabilization is limited because mutations also entail sensitivity to (an) unidentified proteolytic system(s). This observation is consistent with the idea that Fos-expressing viruses have evolved gene expression controls that avoid high protein accumulation-linked apoptosis.

Animals↗

PubMed2002

Multiple degradation pathways for Fos family proteins.

c-Fos protooncoprotein is a short-lived transcription factor with oncogenic potential. It is massively degraded by the proteasome in vivo under various experimental conditions. Those include consititutive expression in exponentially growing cells and transient induction in cells undergoing the G0/G1 phase transition upon stimulation by serum. Though there is evidence that c-Fos can be ubiquitinylated in vitro, the unambigous demonstration that prior ubiquitinylation is necessary for degradation by the proteasome in vivo is still lacking. c-Jun, one of the main dimerization partners of c-Fos within the AP-1 transcription complex, is also an unstable protein. Its degradation is clearly proteasome dependent. However, several lines of evidence indicate that the mechanisms by which it addresses the proteasome are different from those operating on c-Fos. Moreover, genetic analysis has indicated that c-Fos is addressed to the proteasome via pathways that differ depending on the conditions of expression. c-Fos has been transduced by two murine osteosarcomatogenic retroviruses in mutated forms, which are more stable and more oncogenic. The stabilization is not simply accounted for by simple deletion of one of the main c-Fos destabilizers but, rather, by a complex balance between opposing destabilizing and stabilizing mutations. However, although viral Fos proteins have acquired full resistance to proteasomal degradation, stabilization is limited because the mutations they have accumulated, during or after c-fos gene transduction, confer sensitivity to an unidentified proteolytic system(s). This observation is consistent with the idea that fos-expressing viruses have evolved expression machineries to ensure controlled protein levels in order to maintain an optimal balance between prooncogenic and proapoptotic activities of v-Fos proteins.

Amino Acid Sequence↗