>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa human protein known to play an essential role in mRNA 3’-end processing, as well as functioning as an E3 ligase to catalyze ubiquitination and suppression of p53 and other cancer-associated proteins. A RBBP6 knockout mouse model previously suggested that RBBP6 cooperates with MDM2 in polyubiquitinating p53, but is not able to ubiquitinate p53 without the assistance of MDM2. However, unpublished studies from our laboratory suggest that the N-terminal 335 residues of RBBP6, known as R3, are able to ubiquitinate p53 in full in vitro assays, and that the isolated RING finger of RBBP6 is able to catalyse ubiquitination of itself, a phenomenon known as auto-ubiquitination. It is, however, possible that other domains within RBBP6, in particular the ubiquitin-like DWNN domain situated near to the RING finger, may modulate the autoubiquitination and substrate-ubiquitination potentials of the complete protein. / 2022

The tripartite motif (TRIM) family comprises at least 80 members in humans, with most having ubiquitin or SUMO E3 ligase activity conferred by their N-terminal RING domain. TRIMs regulate a wide range of processes in ubiquitination-or sumoylation-dependent manners in most cases, and fewer as adaptors. Their roles in the regulation of viral infections, autophagy, cell cycle progression, DNA damage and other stress responses, and carcinogenesis are being increasingly appreciated, and their E3 ligase activities are attractive targets for developing specific immunother-apeutic strategies for immune diseases and cancers. Given their importance in antiviral immune response, viruses have evolved sophisticated immune escape strategies to subvert TRIM-mediated mechanisms. In this review, we focus on their regulation of IFN-I-mediated innate immune response, which plays key roles in antiviral and antitumor defense.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23605号 / 医博第4792号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長尾 美紀, 教授 竹内 理, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The GPS2 protein (G-protein pathway suppressor 2) is a product of the mammalian gps2 gene. It was originally identified and characterized in the context of G protein mitogen-activated protein kinase (MAPK) signaling pathways. Several studies have linked GPS2 with the inhibition of the ubiquitin conjugating enzyme UBC13. GPS2-mediated inhibition of UBC13 regulates several metabolic and inflammatory pathways. It has been shown that a lack of GPS2 is correlated with an increase in adiposity and inflammation due to the aberrant activity of UBC13 affected pathways. Therefore, understanding the relationship between UBC13 and GPS2 will provide further understanding of the molecular processes involved in adipose tissue levels, inflammation and downstream molecular responses. In this study, we attempt to determine the molecular determinants of GPS2 interaction with UBC13 by optimizing the protein expression protocol required to produce GPS2 protein expression in Escherichia coli in quantities viable for biochemical and structural assays. Our results indicate that optimization of the gps2 sequence is required for efficient GPS2 protein expression in E. coli cells. Thanks to this optimization we have been able to successfully express GPS2 full length and several fragments, however, further optimization will be required for assessing GPS2-UBC13 molecular binding via in vitro binding assays.

The Epstein-Barr virus (EBV) protein LMP1 serves as a paradigm that engages complicated ubiquitination-mediated mechanisms to activate multiple transcription factors. p62 is a ubiquitin sensor and a signal-transducing adaptor that has multiple functions in diverse contexts. However, the interaction between p62 and oncogenic viruses is poorly understood. We recently reported a crucial role for p62 in oncovirus-mediated oxidative stress by acting as a selective autophagy receptor. In this following pursuit, we further discovered that p62 is upregulated in EBV type 3 compared to type 1 latency, with a significant contribution from NF-kB and AP1 activities downstream of LMP1 signaling. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination and activation. As expected, short hairpin RNA (shRNA)-mediated knockdown (KD) of p62 transcripts reduces LMP1-TRAF6 interaction and TRAF6 ubiquitination, as well as p65 nuclear translocation, which was assessed by Amnis imaging flow cytometry. Strikingly, LMP1-stimulated NF-kB, AP1, and Akt activities are all markedly reduced in p622/2 mouse embryo fibroblasts (MEFs) and in EBV-negative Burkitt’s lymphoma (BL) cell lines with CRISPR-mediated knockout (KO) of the p62-encoding gene. However, EBV-positive BL cell lines (type 3 latency) with CRISPR-mediated KO of the p62-encoding gene failed to survive. In consequence, shRNA-mediated p62 KD impairs the ability of LMP1 to regulate its target gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of lymphoblastic cell lines (LCLs). These important findings have revealed a previously unrecognized novel role for p62 in EBV latency and oncogenesis, which advances our understanding of the mechanism underlying virus-mediated oncogenesis. IMPORTANCE As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-kB activation, which involves the ubiquitin machinery, in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-kB is an open question. In this study, we provide evidence that p62 is upregulated in EBV type 3 latency and, in turn, p62 mediates LMP1 signal transduction to NF-kB, AP1, and Akt by promoting TRAF6 ubiquitination and activation. In consequence, p62 deficiency negatively regulates LMP1-mediated gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of LCLs. These important findings identified p62 as a novel signaling component of the key viral oncogenic signaling pathway.

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

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Circadian rhythms are self-sustained, 24h, biological oscillatory processes that are present in organisms ranging from bacteria to human. Circadian rhythms, which can be synchronized by external cues, are important for organisms to adjust their behavior, physiological activity, and metabolic reactions to changes in environmental conditions. Another well-established oscillatory mechanism that shares common organizational and regulatory features with the circadian system, is the cell division cycle. Recent findings reveal that some essential regulators are common to both the cell cycle and the circadian clock. The first half of my thesis (Chapter 2-3) focuses on the function of Period 2 (Per2), a key regulatory component of the negative feedback arm of the clock and tumor suppressor protein, as a modulator of cell cycle response. We found that Per2 binds the C-terminus end of the tumor suppressor p53 thus forming a trimeric complex with p53's negative regulator Mdm2 and preventing Mdm2-mediated p53's ubiquitination and degradation. Thus, Per2 stabilizes p53 under unstressed conditions allowing for basal levels of the protein to exist and be available for a rapid response to take place in case of any stressed signals. Our experiments prove that Per2 plays an indispensible role in p53 signaling pathway. The second half of my thesis (Chapter 4-5) focuses on how Mdm2 and Per2 interplay regulate Per2 availability and its impact on circadian clock function. My research found that Mdm2 targets Per2 for ubiquitination as Mdm2 depletion stabilizes Per2 and, conversely, Mdm2 ectopic expression shorten Per2's half-life. Accordingly, association of Per2 to Mdm2 maps C-terminus of the p53 binding region in Mdm2 and thus, the RING domain remains accessible. Next, we tested the hypothesis that Mdm2-dependent ubiquitination of Per2 directly impacts circadian clock period length. Accordingly, addition of sempervirine nitrate (SN), a specific molecular inhibitor of Mdm2, to MEF cells abrogated Per2 ubiquitination leading to the accumulation of a stable pool of Per2. By recording the oscillatory behavior of the Per2:Luc reporter system in MEF cells treated with SN at different circadian times, we found that inhibition of Mdm2 E3 ligase activity promoted phase advance only when treatment took place during the degradation period. This is in agreement with our findings that radiation, but not light pulses, causes the same phase behavior. Considering the established role of both Mdm2 and p53 in the response of cells to genotoxic stress and Per2 in modulating the clock, the existence of the Mdm2-Per2-p53 complex opens the possibility of various stimuli triggering regulatory mechanisms converging in a critical node. Overall, our work provides a holistic view of how signals are integrated at multiple levels to ensure that environmental signals are sense and responses triggered timely. / Ph. D.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Yes / Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington's disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling. / This work is funded by a Welcome Trust Investigator Award (103844), a Lister Institute of Preventative Medicine Fellowship (137661) and a UKIERI grant (DST/INT/UK/P-147/2016) to S.F.E.- K. JG is additionally funded by a Clinical PhD Fellowship from the Pathological Society of Great Britain and Ireland and the Jean Shanks Foundation (JSPS-CPHD-2018-01).