Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Targeted protein degradation plays a central role in eukaryotic cell regulation and homeostasis. The ubiquitin proteasome system (UPS) is involved in a large number of cellular events, being, day by day, more difficult to find pathways without links with the system. Proteins are marked for degradation by ubiquitin ligases that append polyubiquitin signals, which can recruit the targeted protein to the 26S proteasome for degradation. In the process, polyubiquitin chains appended to the target protein are disassembled into free monoubiquitin for subsequent rounds of substrate tagging by means of deubiquitinating enzymes. Rpn10 is a proteasome receptor that recognizes the Lys48-linked polyubiquitin degradation signal by means of a ubiquitin-interacting motif (UIM). Mutation of the UIM domain of Rpn10 significantly ablates the proteolytic capacity of the proteasome. Rpn10 also contains a Von Willebrand factor A (VWA) domain which is responsible of Rpn10 interactions inside the proteasome. Rpn10 is in equilibrium with a pool of free protein so that it functions as both, a receptor at the proteasome, and an extraproteasomal adaptor. In the present work, we have shown that Rpn10 is monoubiquitinated (Rpn10-mUb) in vivo and that Rpn10-mUb is found in both proteasomal and non-proteasomal pools. Levels of Rpn10-mUb are regulated in vivo by Rps5, a NEDD4 ubiquitin-ligase protein family, and Ubp2, a deubiquitinating enzyme. Our observations link for first time monoubiquitin signal with proteasome regulation. Monoubiquitination strongly inhibits Rpn10 to interact with ubiquitin conjugates by a specific interaction in cis between Rpn10 UIM domain and the linked monoubiquitin. By means of genetic and proteomics tools we found that four sites of Rpn10 sequence are subjected to monoubiquitination by Rsp5 (Lys71, Lys84, Lys99 and Lys268), but Lys84 within its VWA domain is the preferred one. Ubiquitination of Rpn10 inhibits its ability to bind polyubiquitinated substrates, thus functioning as mechanism of inactivation of this receptor. Interestingly, our findings suggest that Rpn10 monoubiquitination could decrease proteasome activity. The UIM of Rpn10 also strongly interacts with the ubiquitin-like domain (Ubl) of Dsk2, a polyubiquitin-binding protein. Furthermore, extraproteasomal Rpn10 plays a critical role in filtering Dsk2 and its substrates from the proteasome. We evaluated the affinity of several forms of Rpn10-mUb to Dsk2 and found that Ub-UIM interaction in cis of Rpn10-mUb impairs the binding of Dsk2 in trans. These results suggest that in an extraproteasomal context, Rpn10 monoubiquitination could regulate the Dsk2-Rpn10 interaction. Notably, the proteasomal pool of Rpn10-mUb is suppressed under perturbations that promote the proteolytic pathway, such as stress by temperature and oxidative stress, whereas the extraproteasomal pool remains fairly constant. To assess whether the distinct behavior of the Rpn10-mUb pools correlated with the modification of different lysine residues of Rpn10, we set up assays of absolute quantification of the two major ubiquitination sites, Lys84 and Lys268, by mass spectrometry (AQUA). The obtained results clearly indicate that Lys84 is the main target of Rsp5 ligase in both pools of Rpn10, enhancing the role of the VWA domain in Rpn10 ubiquitination. Finally, we have combined ubiquitin remnant profiling with quantitative proteomic approaches to identify ubiquitinated species that are increased in RPN10-RAD23 deletion, under standard growth conditions and cold-shock stress. Enriched conjugates have been distributed across different biological functions being proteins involved in RNA processing and transport metabolism accounting for the biggest fractions. Further molecular biology approaches and informatic analysis are required to address which proteins are likely to be true UPS substrates as opposed to proteins regulated by ubiquitination in a non-proteolytic manner. Overall, our results provide a novel evidence of involvement of monoubiquitin signals in the regulation of the availability of substrates to bind proteasomal surfaces. Considering that substrate binding is the first event in the multicatalytic process promoted by the proteasome, the control of the accessibility of proteasome receptors is a crucial level of proteasome regulation. Thus, if the NEDD4 enzyme catalyzed monoubiquitination of Rpn10 inhibits proteasome activity, the control of this reaction could be an interesting target. Classical proteasome inhibitors, such as Bortezomib, which target the proteolytic activity of the core particle of the proteasome, are used as anticancer drugs. We propose that Rpn10 monoubiquitination could be considered as a new target in cancer research. / La degradació de proteïnes juga un rol essencial en la regulació i homeostasi de les cèl•lules eucariotes. Les proteïnes són marcades per a la seva degradació per ubicuitina lligases les quals annexen cadenes de poliubicuitina enviant de manera específica la proteïna al 26S proteasoma on serà degradada. Rpn10 és un receptor del proteasoma que reconeix cadenes d’ubicuitina a través del seu motiu UIM (ubiquitin-interacting motif). Rpn10 també conté un domini VWA el qual és responsable de les interaccions de Rpn10 en el seu context proteasomal. Rpn10 també es troba en equilibri amb una fracció no proteasomal. En el present treball hem demostrat que Rpn10 està monoubicuitinat (Rpn10-mUb) in vivo i que aquesta modificació es troba en ambdues fraccions proteasomal i extraproteasomal. Els nivells de Rpn10-mUb estan regulats in vivo per Rsp5, una ubicuitina lligasa de la família de les NEDD4, i Ubp2, una deubicuitinasa. Mitjançant mètodes genètics i de proteòmica es van trobar quatre lisines modificades per ubicuitina en la seqüència de Rpn10 (Lys71, Lys84, Lys99 and Lys268) éssent la Lys84 la diana preferent de Rsp5. La monoubicuitinació inhibeix la capacitat de Rpn10 d’unir substrats poliubicuitinats i en, conseqüència, és un mecanisme d’innactivació d’aquest receptor. També s’ha trobat que Rpn10-mUb disminuiex l’activitat del proteasoma. Els nostres resultats relacionen per primera vegada la monoubicuitinació amb la regulació del proteasoma, procés que imita la droga Bortezomib. Es proposa que Rpn10-mUb podria considerar-se com a nova diana en la teràpia contra el càncer.

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Due to the action of endogenous and exogenous agents, DNA is subject up to 70,000 lesions per day, thus the existence of repair mechanisms and enzymes is more than necessary. We know basic mechanisms of several specific DNA repair pathways, of which the Fanconi anaemia (FA) repair pathway is one of the least explored. FA is a rare, autosomal recessive disorder characterized by early onset bone marrow failure, developmental defects, genomic instability and predisposition to acute myeloid leukaemia and solid tumours. The primary diagnosis of FA is a hypersensitivity to cross-linking agents of DNA due to inactivation of one of the 21 genes from the FA repair pathway, the so-called FANC genes (FA complementation group). The molecular defect in FA is an impaired repair of DNA interstrand cross-links (ICLs). The ICLs are cytotoxic lesions that inhibit the process of DNA replication and transcription. A crucial step in the FA pathway that initiates ICL repair is a monoubiquitination of FANCD2. FANCD2 monoubiquitination is a base for the recruitment of additional proteins that coordinate DNA repair. Ubiquitin is recruited via activating enzyme E1 (UBA1), ubiquitin-conjugating enzyme E2T (UBE2T) and transferred onto FANCD2 by multisubunit E3 ligase (FA core complex). There are up to 11 different proteins...

Fanconi anemia is an autosomal recessive disorder caused by mutation in one of Fanconi genes and it is manifested by developmental abnormalities, bone marrow failure, predisposition to cancer, cellular sensitivity to cross-linking agents and many other symptoms. Proteins encoded by Fanconi genes and some other proteins are part of Fanconi anemia pathway (FA pathway), which is responsible for DNA repair of an interstrand cross-link (ICL). The repair by this pathway requires monoubiquitination of FANCD2, which is induced and regulated by ATR dependent FANCI phosphorylation. The FANCI phosphorylation initiates the FA pathway but the molecular mechanism of this initialization is not known. Furthermore the proper function of entire pathway requires both: sequence of phosphorylation events of FANCI and monoubiquitination of FANCI:FANCD2 complex . The principle of this work was to study molecular mechanism of initiation and regulation of FA pathway by FANCI phosphorylation. Therefore phosphomimetic mutants of FANCI have been created to investigate their role in processes leading to FANCD2 monoubiquitination. The main aim was to reveal how the phosphorylation of FANCI affects DNA binding and also DNA binding of FANCI:FANCD2 complex. Since both DNA and FANCI phosphorylation are required for proper FANCD2...

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