Cdc48A and its human homologue P97 are from ATPase family, which play a variety of roles in cellular activates and it has a crucial involvement in protein quality control pathways. It is best known for its involvement in endoplasmic reticulum associated protein degradation (ERAD), where it mediates the degradation of the aggerated or misfolded proteins by the proteasome. Considering the multiple functions of Cdc48A in many protein regulatory processes, it is a potential therapeutic target for neurogenerative diseases and cancer. Cdc48A polypeptide comprises N domain, followed by D1 and D2 domains respectively that are joined by linkers, whereas functionally it forms a homo hexameric complex. Since Cdc48A is from the ATPase family, it uses the ATP hydrolysis to generate a mechanical force with its co-factors to perform its functions. There are many cofactors that interact with Cdc48A and two of them are Ufd1-NpI4 which in turn interact with ubiquitinated proteins from the ER membrane. The mechanism linking the conversion of the energy of ATP hydrolysis into mechanical force and unfolding the substrate is vague. My aim is to reconstruct a first 3D- model of plant Cdc48A using single particle cryo-EM, which serves the basis to conduct more detailed mechanistic studies towards substrate unfolding and threading/unfolding in the future. In general, results showed one defined structure of cdc48A at ~ 9.8 Å, which is the ADP-ATP conformation. Although another other structure was also resolved at ~ 8.9 Å, it was hard to characterize due to its dissimilarity with known structures of Cdc48A homologues and thus requires further characterization.

p97 is a member of the AAA family of proteins (ATPase Associated with various cellular Activities). It is a highly conserved and abundant protein and functions in numerous ubiquitin-mediated processes including ERAD. Endoplasmic Reticulum Associated Degradation is the process by which misfolded/ubiquitinated proteins translocate out of the ER and migrate to the proteasome for degradation. p97 maintains substrate misfolding and mediates its exit from the ER and trafficking to the 26S proteasome. It also plays important roles in protein trafficking, the cell-cycle, apoptosis and homeotypic Golgi Apparatus and Endoplasmic Reticulum membrane fusion after mitosis. In addition, p97 plays a role in the aggresome-autophagy degradation pathway, which handles the ubiquitin-mediated destruction of aggregate-prone, misfolded, cytosolic proteins. p97 mutation is the causative alteration in the disorder, IBMPFD, which is marked by defects in autophagy. This broad diversity of function is mediated through p97's interaction with a large group of adaptor proteins. Many of these adaptors harbor both p97 interaction motifs and ubiquitin association domains. However, more than half of known p97 adaptors do not. Their function is largely unknown. UBXD1 is one known adaptor for p97 that does not have a ubiquitin association domain (UBA), and has been shown to have decreased interaction with IBMPFD mutant p97R155H and p97A232E. Recently, it has been suggested to perform a role in protein trafficking, specifically in monoubiquitinated caveolin-1 internalization and trafficking to the endosome. A novel high abundance UBXD1 interacting partner has been identified via solution-based mass spectrometric analyses. ERGIC-53, the namesake of the ER-Golgi Intermediate Compartment, has been shown to be involved in bi-directional trafficking between the ER and Golgi. The association between UBXD1 and ERGIC-53 is unique among UBX family members. Deletional analysis has shown that unlike p97, the ERGIC-53-UBXD1 interaction takes place in the extreme amino terminus of UBXD1, (within the first 10 amino acids) which is predicted by computer modeling to form a hydrophobic binding pocket. Further site-directed mutagenesis work has clearly shown four amino acids (3 highly hydrophobic) are crucial for maintaining this interaction. They have been modeled to form a conserved alpha-helix. ßCOPI, a primary member of the COPI coatomer complex which is involved in protectively coating ERGIC-53 positive vesicles, is also thought to be involved with the ERGIC-53-UBXD1-p97 pathway. ßCOPI has been identified as a UBXD1-independent interactor with p97. Modest UBXD1 over- expression using a ponasterone inducible system has shown that UBXD1 modulates ERGIC-53 localization. Additionally, a functional link between UBXD1, p97 and ERGIC-53 in autophagy has been discovered through the use of a highly efficient, miR30-based, inducible knockdown system. Upon individual knockdown of UBXD1, p97 and ERGIC-53, autophagic markers p62 and LC3-II accumulate at relatively high levels in normal culture conditions, strongly suggesting a role in mediating basal autophagy. However, when placed under starvation conditions, autophagy progresses and p62 is degraded. It is speculated from these studies that a p97/UBXD1 complex plays a role in regulating the trafficking of ERGIC-53 positive vesicles and this activity plays an important role in autophagy. / Molecular Biology and Genetics

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23553号 / 生博第464号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 井垣 達吏, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The Fragile X-related mental retardation 1 (FXR1) is an N6-Methyladenosine reader involved in mRNAs metabolism like mRNA splicing, stability, transport, and miRNA regulation. It is also important in transcription, cell proliferation, differentiation, translation, polysome assembly and stress granule assembly. The protein is present in all eukaryotic cells, but so far it has been specifically essential for correct neural function. Until now, FXR1 has not been investigated in the concept of Human Adenovirus infection but we have observed an upregulation of FXR1 during the late phase of the Human Adenovirus 5 (HAdV-5) infection and an upregulation of some late HAdV-5 proteins in HeLa cells overexpressing FXR1. Our results furthermore showed that a FXR1 knockdown resulted in a reduced level of some HAdV-5 proteins at the same time as HAdV-5 mRNA were stabilized, indicating that FXR1 might be involved in translation of HAdV-5 late genes. Further investigation of the mechanism behind FXR1 mediated translation, a Death-associated protein 5 (DAP5) was founded to have an overall effect on the translation of HAdV-5 late proteins. / Part of a post-doctoral research

La protéine p97/VCP est un membre de la famille des ATPase AAA+ et joue un rôle majeur dans de nombreux processus cellulaires tel que le contrôle de l’homéostasie protéique ou de fonctions associées à la chromatine (transcription, réplication, dommage à l’ADN, progression du cycle cellulaire). De plus, la protéine p97/VCP est impliquée dans un nombre croissant de maladies dont les cancers où il a été montré qu’elle contribue à l’homéostasie protéique et l’adaptation au stress oncogéniques. En effet, l’expression de la protéine p97/VCP est augmentée dans de nombreux cancers et dans certains cas corrèle avec une récurrence de la tumeur et un mauvais pronostique pour les patients. Cependant, le mécanisme moléculaire précis par lequel la protéine p97/VCP régule l’homéostasie protéique des cellules tumorales reste incertain. Pour remédier à cela, nous avons démontré un rôle de la protéine p97/VCP dans le contrôle de l’expression des gènes lors du stress du Réticulum Endoplasmique (RE). Nous avons trouvé que en conditions basales, la protéine RuvBL2 fait partie d’un complexe remodeleur de la chromatine qui contient les protéines HDAC1 et mSin3A et agit comme un répresseur des gènes de stress du RE. De plus, nous avons identifié le gène Gli1, un effecteur connu de la voie de signalisation Hedgehog comme cible de la protéine p97/VCP et du complexe RuvBL2-HDAC1-mSin3A. Ainsi en condition de stress du RE, la voie de signalisation Hedgehog qui a été impliqué dans le développement de cancers est activée. Globalement, nos travaux indiquent que p97/VCP agit comme un interrupteur moléculaire pour inactiver le complexe répresseur RuvBL2-HDAC1 en condition de stress du RE et ainsi activer les gènes de stress du RE et de la voie de signalisation Hedhehog de façon non-canonique. / P97/VCP is a member of the AAA+ ATPase family that plays major roles in various cellular processes including control of protein homeostasis and chromatin-associated functions (transcription, replication, DNA damage, cellular cycle progression). Moreover, p97/VCP is involved in a growing number of diseases including cancers in which it has been shown to contribute to protein homeostasis and adaptation to oncogenic stresses. Indeed, p97/VCP expression is increased in numerous cancers and in some cases correlates with tumor recurrence and poor prognosis for patients. However, the precise mechanism by which p97/VCP regulates tumor cell proteostasis remains unclear. To address this, we demonstrated a role of p97/VCP in gene expression control upon endoplasmic reticulum (ER) stress. We found that in basal conditions, RuvBL2 is part of chromatin remodeler complex that included HDAC1 and mSin3A and act as a repressor of ER stress genes. However under ER stress, ubiquitinylated RuvBL2 is degraded by p97/VCP thus causing activation of ER stress genes. Moreover, we have identified GLI1, a known effector of Hedgehog signaling, as a target of the p97/VCP and RuvBL2-HDAC1-mSin3A complex. As a result under ER stress conditions, the Hedgehog pathway which have been linked to cancer development is non-canonically activated. Overall, our work indicated that p97/VCP acts as a molecular switch to inactivate RuvBL2-HDAC1 repressor complex under ER stress thus activating ER stress genes and Hedgehog genes in a non-canonical manner.

Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation ( the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system ( UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.

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

Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.

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