Isabelle Jariel-Encontre

Ubiquitin-independent degradation of proteins by the proteasome.

The proteasome is the main proteolytic machinery of the cell and constitutes a recognized drugable target, in particular for treating cancer. It is involved in the elimination of misfolded, altered or aged proteins as well as in the generation of antigenic peptides presented by MHC class I molecules. It is also responsible for the proteolytic maturation of diverse polypeptide precursors and for the spatial and temporal regulation of the degradation of many key cell regulators whose destruction is necessary for progression through essential processes, such as cell division, differentiation and, more generally, adaptation to environmental signals. It is generally believed that proteins must undergo prior modification by polyubiquitin chains to be addressed to, and recognized by, the proteasome. In reality, however, there is accumulating evidence that ubiquitin-independent proteasomal degradation may have been largely underestimated. In particular, a number of proto-oncoproteins and oncosuppressive proteins are privileged ubiquitin-independent proteasomal substrates, the altered degradation of which may have tumorigenic consequences. The identification of ubiquitin-independent mechanisms for proteasomal degradation also poses the paramount question of the multiplicity of catabolic pathways targeting each protein substrate. As this may help design novel therapeutic strategies, the underlying mechanisms are critically reviewed here.

Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo?

The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanims responsible for the selective destruction of proteins within cells.

Complex mechanisms for c-fos and c-jun degradation.

Abstractc-fos and c-jun proto-oncogenes have originally been found in mutated forms in murine and avian oncogenic retroviruses. They both define multigenic families of transcription factors. Both c-jun and c-fos proteins are metabolically unstable. In vivo and in vitro work by various groups suggests that multiple proteolytic machineries, including the lysosomes, the proteasome and the ubiquitous calpains, may participate in the destruction of c-fos and c-jun. The relative contribution of each pathway is far from being known and it cannot be excluded that it varies according to the cell context and/or the physiological conditions. It has been demonstrated that, in certain occurrences, the degradation of both c-fos and c-jun by the proteasome in vivo involves the ubiquitin pathway. However, the possibility that proteasomal degradation can also occur in a manner independent of the E1 enzyme of the ubiquitin cycle remains an open issue.

A Novel Role for PA28γ-Proteasome in Nuclear Speckle Organization and SR Protein Trafficking

In eukaryotic cells, proteasomes play an essential role in intracellular proteolysis and are involved in the control of most biological processes through regulated degradation of key proteins. Analysis of 20S proteasome localization in human cell lines, using ectopic expression of its CFP-tagged alpha7 subunit, revealed the presence in nuclear foci of a specific and proteolytically active complex made by association of the 20S proteasome with its PA28gamma regulator. Identification of these foci as the nuclear speckles (NS), which are dynamic subnuclear structures enriched in splicing factors (including the SR protein family), prompted us to analyze the role(s) of proteasome-PA28gamma complexes in the NS. Here, we show that knockdown of these complexes by small interfering RNAs directed against PA28gamma strongly impacts the organization of the NS. Further analysis of PA28gamma-depleted cells demonstrated an alteration of intranuclear trafficking of SR proteins. Thus, our data identify proteasome-PA28gamma complexes as a novel regulator of NS organization and function, acting most likely through selective proteolysis. These results constitute the first demonstration of a role of a specific proteasome complex in a defined subnuclear compartment and suggest that proteolysis plays important functions in the precise control of splicing factors trafficking within the nucleus.

Fos family protein degradation by the proteasome

c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

[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.

Le catabolisme protéique intracellulaire : une fonction biologique majeure. Partie I : les mécanismes de dégradation.

La degradation des proteines est une fonction intracellulaire majeure. En premier lieu, elle assure le «menage cellulaire» et permet ainsi la survie des cellules en empechant laccumulation de peptides toriques. Elle est aussi impliquee dans le maintien de lhomeostasie cellulaire, la genese des peptides antigeniques dans le cadre de la reponse immunitaire, la regulation detapes cles du developpement embryonnaire et du gicle cellulaire. Les bases biologiques et biochimiques du catabolisme proteique sont encore peu ou mal connues. Quelques reponses fragmentaires ont ete apportees concernant le nombre de voies cataboliques operant dans une cellule, le nombre de ces voies agissant sur une meme proteine, le(s) site(s) de degradation des proteines evoluant dans differents compartiments cellulaires, les motifs reconnus par les systemes proteolytiques et la regulation du processus de degradation

Different catabolic pathways are responsible for c-JUN and c-FOS proteins degradation in embryo fibroblasts stimulated for growth

Major events in early development are associated with zygotic activation and cell cycle control in a multicellular organism. In Xenopus, following fertilization, the embryo enters a succession of rapid and synchronous divisions during which transcription is barely detectable. Progressively, cell cycles lengthin, become asynchronous, while the zygotic genbme becomes transcriptionally competent (Newport J., Kirschner M. (1982a), Cell, 30, 687-96 ; Newport I., Kirschner M. (1982b),Cell 30, 675-86). Changes in chromatin composition during that time period were observed previously (Dimitrov S., Almouzni G., Dasso M., Wolffe A. P. (1993), Dev. Biol., 160, 214-227 ; Almouzni G., Khochbin S., Dimitrov S., Wolffe A. P. (1994). Dev. Biol., 165, 654-669). Of interest to the present study, one of the major transition concerned the acetyiation state of the N-terminal tail of histone H4, a modification often associated with transcriptional activity (Turner B. M. (!993), Cell. 75, S-8). We examined whether these changes were associated with remodeling of nuclear organization within the embryonic cells. We developed a method for ifolaiion of nuclei ai different deveIopmental stages. We verified that we were able to preserve their functional properties. This included testing specific nuclear Import, replication and transcription (for transcriptionally competent nuclei). Starting from this material, we analysed by immunolocalisation the distribution of specitic markers. We made use of specific antibodies directed against the various acetylated forms of histone H4 (Turner B. M., Fellows G. (1989). Eur. J. Biocllem., I79, 13 1139) and against the major subunit of RNA polymerase II (Thompson N. E., Aronson D. B., Burgess R. (1990). J. Biol. Chem., 265, 7069-7077). For each marker, we observed distinct .pattems of subnuclear localization. These patterns evolved in parallel with the developmental stages. These observations suggest that specific mechanisms are involved in the establishment of functional chromatin domains in the nucleus during early development. EVIDENCES FOR A “CROSS-TALK” UETWEEN REFINOIC AC:II> RECEPTORS AND MYOD IN MUSCLE CELLS

Le catabolisme protéique intracellulaire : une fonction biologique majeure. Partie II : exemples de dégradation conditionnelle et genèse des peptides antigéniques

Le catabolisme proteique joue un role majeur dans la cellule, et ne se contente pas dassurer lelimination des proteines dont laccumulation deviendrait toxique. Certaines degradations sont finement reglees, en particulier celle des facteurs de differenciation et de transcription, des proteines onco-suppressives (p53) et des cyclines mitotiques. Le catabolisme proteique est donc implique dans le controle detapes cles de la differenciation et du cycle cellulaire. En outre, il est a la base de la reponse immune specifique en permettant lappretement de peptides antigeniques.