Rozprawy doktorskie na temat „E3 LIGASE ACTIVITY”

Half of cancers retain wild type p53 but have alterations in the pathways involved in p53 regulation. Murine double minute 2 (Mdm2) regulates p53 by acting as an E3 ubiquitin ligase, which tags p53 for degradation through the proteasome. A small molecule inhibitor, a 5-deazaflavin analogue, has previously been identified by high throughput screening to inhibit Mdm2 E3 ubiquitin ligase activity, thereby reactivating apoptotic function of p53 selectively in cancer cells. Ninety 5-deazaflavin analogues have been synthesised by an optimized existing method and a novel method of synthesis, using the required 6-anilinouracil and 2-p-toluenesulfonyloxybenzaldehyde.The biological ability of the 5-deazaflavin analogues to act as inhibitors of Mdm2 E3 ubiquitin ligase activity to reactivate p53 has been ascertained. A new quantitative biological assay was developed, by scientists based at the Beatson Institute, for 5-deazaflavin compounds, showing excellent inhibition of Mdm2 E3 ubiquitin ligase activity on the previous qualitative biological assay, to yield IC50 data. The biological results have established a clear and logical structure-activity relationship comprising of an electron-withdrawing hydrophobic substituent at the nine position and the N10 phenyl being a prerequisite for activity as a Mdm2 inhibitor. Also meta substitution of the N10 phenyl improves activity against Mdm2 E3 ubiquitin ligase activity. Hit optimization has occurred with 10-(3-chlorophenyl)-9-trifluoromethyl-5-deazaflavin being thirty times more active than the previous identified hit compound, 10-(4-chlorophenyl)-7-nitro-5-deazaflavin. Using the X-ray crystal structure of the Mdm2/MdmX heterodimer, an improved understanding of how Mdm2 acts as an E3 ubiquitin ligase is described and used to form a hypothesis of how 5-deazaflavin analogues function as inhibitors of Mdm2. The work suggests the principle that small molecular weight compounds can inhibit E3 ubiquitin ligases as a possible anti-cancer therapy, and provide the foundation and framework for additional studies and investigation in a new and developing field of medicinal chemistry.

TRIM proteins are a family of ubiquitin E3 ligase enzymes characterized by the presence of a conserved N-terminus, the tripartite motif, which consists of a RING finger domain, two B-box motifs and an alpha-helical Coiled-coil region (RBCC). We focused our attention on TRIM18/MID1, the gene responsible for the X-linked form of Opitz G/BBB syndrome, a congenital disease characterized by defects in midline development and mental retardation. More recently, MID1 has also been found as overexpressed in some aggressive prostate cancers. The role of MID1 within the cell and the target(s) of its E3 ubiquitin activity during cellular processes are still not completely unravelled. In order to better investigate MID1 function and to find new cellular partners for this protein a two hybrid assay was performed in our laboratory. By means of this screening, BRCA2-Associated Factor 35 (BRAF35 or HMG20b) was identified as a novel MID1 interacting protein. BRAF35 is a non-canonical High-Mobility-Group (HMG) protein that has a role in both neuronal differentiation and in cell cycle progression. Moreover BRAF35 sumoylation has been shown to be fundamental for its antineurogenic activity and is inhibited by the interaction with its homologue iBraf. The aim of the project was to characterise the functional role of MID1/BRAF35 interaction and to understand if MID1, as an E3 ubiquitin ligase, regulates BRAF35 during cytokinesis. The first evidence obtained from the preliminary screening was confirmed through MBP pull-down assay and co-immunoprecipitation, identifying the coiled-coil region of both proteins as responsible for the binding. We further investigated on a possible regulation of BRAF35 by the ubiquitin proteasome system and we recognized BRAF35 as a poly-ubiquitinated protein and we found that its abundance is regulated in a proteasome-dependent manner. In addition, overexpression of MID1 or its domain-deleted mutants altered BRAF35 stability and post-translational modification suggesting a MID1-dependent BRAF35 ubiquitination that implicates also K63-polyUb dependent signalling involvement. Additionally, we found that MID1 and BRAF35 colocalize not only during interphase but also at the intercellular bridge during cytokinesis. Consistent with this observation, we observed a cell cycle-related regulation of BRAF35 protein level. Moreover, MID1 depletion rescued the cytokinetic defect caused by BRAF35 silencing, leading to a decrease of binucleation, but promoted a blebbing phenotype in dividing cells. This indicates that a fine regulation of the two proteins for the completion of cytokinesis is required and suggests an additional and new role for MID1 in cytokinesis regulation.

The ubiquitylation cascade regulates multiple cellular functions and is involved in numerous diseases. The distinct transfer cascade, involving E1-E2-E3 enzymes, serves as a promising target for drug development. However, E3 ligases (E3s) represent an important class of enzymes yet there are currently no effective tools for profiling their activity. Herein, a new class of E3 activity-based probe (ABP) is presented which is built by re-engineering ubiquitin (Ub)-charged E2 conjugating enzymes. The utility of these probes has been demonstrated by the rapid dissection of the activation determinants of the RING-Between-RING E3 (RBR) E3, Parkin. Furthermore, biotin-E3 ABPs allow us to systematically discover and dissect the E3 activities of a broad spectrum of E3s that are associated with different diseases. By interfacing the ABPs with mass spectrometry, we establish an activity based protein profiling (ABPP) system and apply it to uncover a new class of E3. We show that MYCBP2 is an E3 ligase with a novel mechanism of action that ubiquitylates threonine residues. MYCBP2 contains a RING domain, that recruits the ubiquitin-loaded E2, and a novel Zn-binding fold that contains two catalytic cysteine residues which relay the Ub to substrate via two thioester intermediates (RING-Cys-Relay, RCR). This discovery demonstrates the power and potential of our E3 activity based protein profiling (ABPP) system.

Ubiquitination is a post-translational modification of proteins with broad regulatory roles across cellular biology. This process involves the addition of ubiquitin molecules on target proteins, altering their cellular role and properties. Ubiquitination is performed by an enzymatic cascade consisting of three enzymes: ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). The tripartite motif (TRIM) family of proteins constitutes one of the largest subfamilies of RING E3 ligases and the majority of them are contributing to the regulation of innate immune responses. They are characterized by a conserved tripartite motif in their N-terminal region which comprises a RING domain, one or two B-box domains and a coiled-coil region. Self-association is believed to be crucial for catalytic activity of TRIM proteins, however, the precise molecular mechanism underlying this observation remains elusive. The work presented in this thesis provides insights into the E3 ligase function of TRIM25 and shows how its oligomeric state is linked to its catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and a ubiquitin-loaded E2 identifies the structural and mechanistic features that promote activation of E2~Ub allowing us to propose a model for the regulation of activity in the full-length protein. In the second part of this thesis, the molecular details of Influenza A NS1-mediated TRIM25 inhibition are presented. The crystal structures of NS1 bound to TRIM25 along with biochemical analysis allowed us to identify the interacting domains and propose a model for the inhibition of substrate ubiquitination during viral infection. The results of this project extend our understanding of the mechanism, structure and regulation of TRIM E3 ligases and their substrates, leading to increased chances of targeting specific steps of the ubiquitination pathway during disease.

E3 ubiquitin ligases are key regulatory enzymes of the ubiquitination pathway as they are responsible for substrate specificity. This thesis aimed at deciphering the molecular mechanisms through which two different E3 ligases, Nedd4 and Rabex-5, exert their activity. Nedd4 is the prototype for HECT-E3 ligase while Rabex-5, containing an A20 zinc finger domain (ZnF_A20) instead of a canonical RING, could be defined as an atypical RING-E3 ligase. In the case of Nedd4, we provided the first crystal structure of the catalytic intermediate of HECTNedd4~Ub in complex with Ub non-covalently bound to the UBD present in the N-lobe of HECTNedd4. Our structure represents the next step of the transfer of UbD from the catalytic cysteine of E2 to the one of E3 in which the UbD C-terminal tail is in an extended conformation primed for catalysis. Our data strongly supports the sequential addition model proposed for HECT proteins. Within this study we also clarified some aspects of Rabex-5 as E3 ligase. By yeast-two-hybrid, GST-pull-down assays and ITC analysis, we identified specific E2 partners, Ube2D and Ube2E families, that bind Rabex-5 only when in their active Ub-loaded state. Performing in vitro auto-ubiquitination assay and disulfide stability assay we confirmed that ZnF_A20 is the minimal domain responsible for the catalytic activity. To obtain the structure of the Rabex-51-74:E2-Ub complex, we tested, unfortunately without success, crystallization trials and SAXS analysis with various samples. We also analyzed Rabex-5 catalytic activity towards on H-Ras, which is the unique substrate of Rabex-5 so far identified, and we disproved that H-Ras is a Rabex-5 substrate. To identify candidate substrates we profiled 20.000 human proteins using a microarray-based ubiquitination screening. A list of 67 proteins represent the most statistically stringent and conservative estimate for Rabex-5 substrates that we are going to validate in the nearest future, starting from the ones involved in the endocytic pathway.

In the eukaryotic genome, DNA and histone modifications regulate chromatin function and mediate basic processes such as gene transcription, DNA repair and DNA replication. Maintaining chromatin modifications after DNA replication is essential for chromatin homeostasis, especially for regions of the genome that need to be kept silenced such as repetitive elements. The maintenance DNA methyltransferase, DNMT1, is responsible for ensuring that cytosine methylation at CpG dinucleotides, and thus proper transcriptional programmes, are propagated to the daughter cells. DNMT1 is specifically recruited to newly replicated, hemi-methylated DNA and the E3-ubiquitin ligase UHRF1 (Ubiquitin-like containing PHD- and RING-finger domains protein 1) plays a critical role for this. The mechanisms of the recruitment of DNMT1 to chromatin via UHRF1 are currently an area of active investigation. Several studies using modified nucleosomes, histone peptides and DNA oligonucleotides have identified UHRF1 to bind to hemi-methylated CpG dinucleotides and to histone H3 di- or tri-methylated at Lys-9. Since UHRF1 was also found to interact with DNMT1, it was postulated that UHRF1 acts as an adapter that directly recruits DNMT1 to newly replicated DNA. Additionally, it has recently been reported that the E3-ubiquitin ligase activity of its C-terminal RING-finger is required for the recruitment of DNMT1 to replication forks. Ubiquitylation of either K18 or K23 on histone H3 that is recognised by a ubiquitin-interacting motif within DNMT1 appears to be critical for DNMT1 targeting but the recruitment mechanism has so far not been completely elucidated. This study has investigated the binding and E3-ubiquitin ligase activity of UHRF1 in the context of physiologically relevant chromatin substrates. Using a fully reconstituted system, the chromatin binding and enzymatic activity of UHRF1 and how this is linked to its intra-molecular arrangement have been elucidated. In the context of modified nucleosome substrates, we observe an increase in binding of recombinant UHRF1 in the presence of hemi-methylated DNA whilst with histone H3K9me2/3, only a small increase in binding is detected. We also provide evidence that binding to nucleosome core particles is enhanced by a basic region between the SRA-domain and the RING-finger. This so called polybasic region or PBR has previously been implicated in the regulation of UHRF1 binding to H3K9me2/3 marks. Our findings therefore suggest that binding of UHRF1 to physiological chromatin substrates is more complex than previously thought. In-solution crosslinking/mass spectrometry experiments using the full-length protein confirm that UHRF1 exhibits complex intra-molecular contacts that can potentially regulate its interaction with chromatin or other factors. In addition to reported contacts between the PBR with the Tandem-Tudor domain and between the PHD-finger and the SRA-domain, the UBL-domain also makes extensive contacts to other regions within UHRF1. These appear to be weak and dynamic. Crucially, removal of the UBL-domain does not affect nucleosome binding but does result in a strong reduction in UHRF1 E3-ubiquitin ligase activity. Further experiments suggest that the UBL-domain is involved in establishing the enzyme/substrate complex between the E2-conjugating enzyme and the chromatin substrate and in stimulating the transfer of ubiquitin from the E2~Ub complex to histone H3. In summary, by combining a crosslinking/mass spectrometry approach to interrogate the intra-molecular arrangement of UHRF1 with fully reconstituted enzyme and chromatin-binding assays using physiologically relevant substrates, we have identified a function for the UBL-domain of UHRF1. Our results suggest that the UBL is highly flexible in solution and that it forms transient contacts with other parts of UHRF1 and the E2-conjugating enzyme that are required for the formation of the E2/E3/substrate complex in allosterically activating ubiquitin transfer from the E2~Ub to the histone target substrate. These findings assign, for the first time, a function for the UBL-domain and pave the way for further investigation of the involvement of this domain in the physiological role of UHRF1.

La voie de signalisation TGF-β joue un rôle biphasique durant la cancérogenèse. Mon laboratoire a identifié une nouvelle protéine inhibitrice de la voie TGF-β, WWP1. WWP1 est une E3 ubiquitine ligase qui induit la polyubiquitination et la dégradation du récepteur de type I au TGF-β. De plus, le gène WWP1 est amplifié dans une large proportion de cancers mammaires et prostatiques, suggérant que WWP1 pourrait jouer un rôle clé dans les processus de cancérogenèse liés au TGF-β. Mon projet de thèse était donc de caractériser la régulation de l’activité catalytique de WWP1 ainsi que son mécanisme d’action dans la cellule. Mes résultats montrent qu’à l’état basal, WWP1 est monoubiquitinée, son activité de polyubiquitination étant réduite par l’effet inhibiteur qu’exercent les domaines C2 et/ou WW sur son domaine HECT. En présence de substrats, la protéine WWP1 « s’ouvre » et peut alors induire la polyubiquitination et la dégradation de ses substrats. De plus, nous avons observé qu’un mutant de WWP1, détecté dans un cancer de la prostate, est incapable de s’autoréguler selon ce modèle. Il présente une plus forte activité ligase envers lui-même et ses substrats, ce qui entraîne une atténuation de la réponse cytostatique du TGF-β pouvant conférer une activité oncogénique à WWP1. De plus, nous avons identifié STARD13 comme un nouveau partenaire de WWP1. STARD13 est une protéine à activité RhoGAP, considérée comme un suppresseur de tumeur. Nous avons montré que STARD13 permet l’association de WWP1 avec la GTPase RhoA, entraînant ainsi la polyubiquitination et la dégradation de RhoA. De façon intéressante, le complexe WWP1/STARD13 est impliqué dans le remodelage de l’architecture du cytosquelette en dégradant préférentiellement la forme activée de RhoA. Ces résultats ont permis d’identifier un nouveau rôle de WWP1 qui pourrait jouer un rôle essentiel durant la migration des cellules cancéreuses lors du processus métastatique. La caractérisation de nouveaux mécanismes de régulation et d’action de WWP1 devrait permettre à terme d’identifier si WWP1 est un marqueur diagnostique dans le cancer et/ou une nouvelle cible thérapeutique pour le développement de médicaments anticancéreux
The TGF-β pathway plays a biphasic role during cancerogenesis. My laboratory identified a new protein, WWP1, as a negative regulator of TGF-β signaling. WWP1 is an E3 ubiquitin ligase that triggers polyubiquitination and degradation of TGF-β type I receptor. A genomic amplification of WWP1 is found in a large portion of mammary and prostatic tumors, suggesting a key role for WWP1 during carcinogenesis related to TGF-β. My thesis project was to determine the regulation of the catalytic activity of WWP1 and a new molecular mechanism of action of WWP1 whose deregulation can be implicated in cancerogenesis. My results indicate that at steady states, WWP1 is monoubiquitinated, its polyubiquitination activity being silenced due to the inhibitory effects of C2 or/and WW domains on its Hect domain. In presence of substrates, WWP1 is « opened » and induces polyubiquitination and degradation of its substrates. Moreover, a WWP1 mutation found in prostate cancer disrupts this regulatory mechanism. It possesses an increased ligase activity towards itself and its substrates, which leads to the attenuation of TGF-β cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1. We also identified STARD13 as a novel WWP1 interacting partner. STARD13 has a RhoGAP activity, and is considered as a tumor suppressor. We have shown that STARD13 mediates the association of WWP1 with the GTPase RhoA, ultimately leading to RhoA polyubiquitination and degradation. Interestingly, the WWP1/STARD13 complex is involved in the actin cytoskeleton rearrangement by preferentially targeting the active form of RhoA for degradation. These results reveal a previously unrecognized role for WWP1, which could play a key role in the migration of cancer cells during metastasis. Characterization of new regulation and action mechanisms for WWP1 should allow identifying whether WWP1 is a diagnosis biomarker in cancer and/or a new therapeutic target for the development of anticancer drugs

Une famille de petites protéines, appelée famille des molécules d'ubiquitine (Ubls), joue un rôle essentiel dans de nombreux aspects de la réponse au stress. Des défauts dans les composants de la famille de l'ubiquitine sont souvent retrouvés dans les pathologies, notamment dans certain type de cancer et les maladies neurodégénératives.L'une des Ubls qui présente le plus d'identité et de similarité avec l'Ubiquitine est NEDD8. NEDD8 fonctionne de manière similaire à Ub mais utilise un mécanisme de conjugaison distinct. La modification de NEDD8 est essentielle au maintien de l'homéostasie de la cellule car elle joue un rôle majeur dans la régulation de la viabilité, de la croissance et du développement. Pour cette raison, de nombreux composants de NEDD8 ont été dérégulés dans de nombreux cancers. NEDD8 peut modifier un large éventail de substrat protéique, et peut également s’auto-modifier entrainant la création de chaînes polyNEDD8. Récemment, la présence de chaînes polyNEDD8 a été liée à la régulation de la mort cellulaire - apoptose et parthanatos. De plus, il a été récemment rapporté que NEDD8 pendant un stress protéotoxique peut être employé par la machinerie de conjugaison Ub. Cela aboutit à la création de chaînes NEDD8 hybrides tels que NEDD8-Ub et NEDD8-SUMO. En effet, des articles récents ont montré que NEDD8 a non seulement la capacité de modifier Ub mais également SUMO-2. La présence des chaînes hybrides NEDD8 a été liée à la création d'agrégats nucléaires formés pendant le stress protéotoxique, ce qui peut jouer un rôle protecteur pendant l'exposition au stress.Connaissant l'importance de la régulation des protéines par la NEDDylation, nous étions également conscients du manque de connaissances sur le mécanisme qui joue un rôle dans la création et la déconjugaison des différentes entités NEDD8. Jusqu'à présent, deux enzymes déNEDDylantes ont été signalées, mais aucune enzyme n'a été testée pour sa capacité à reconnaître et à traiter les chaînes hybrides NEDD8. De plus, nos connaissances sur les ligases E3 responsables de la NEDDylation du substrat, bien qu'en expansion, sont encore très limitées. De plus, étant donné que NEDD8 peut s’auto-modifier via l’utilisation de l’une de ses dix lysines, elle peut générer une très large gamme de signaux par la formation de chaînes polyNEDD8 et hybrides NEDD8. Ces chaines peuvent être reconnues de la même manière que les chaînes polyUb, mais aucune étude ne s'est concentrée sur la détermination de leurs interacteurs jusqu'à présent.Dans ce travail, nous nous sommes concentrés sur l'exploration de ces mécanismes inconnus de conjugaison et de déconjugaison de NEDD8 mentionnées précédemment. En utilisant des approches de biologie chimique, nous avons testé une variété d'enzymes et déterminé que les chaînes polyNEDD8 sont exclusivement traitées par l'enzyme NEDP1. Cependant, la déconjugaison des chaînes hybrides NEDD8 nécessite l'action coordonnée de différentes enzymes de déconjugaison avec une spécificité distincte pour Ub ou SUMO. Nous avons également utilisé des dimères NEDD8-NEDD8 et NEDD8-Ub synthétisés chimiquement afin de rechercher leurs interacteurs et utiliser les données recueillies pour approfondir nos connaissances sur la biologie des chaînes hybrides NEDD8 dans les agrégats nucléaires. En utilisant les sondes NEDD8-Dha, nous avons identifié un groupe de protéines qui sont potentiellement impliquées dans la machinerie de NEDDylation. Par la confirmation biologique des résultats obtenus, nous avons montré que les ARNt ligases - GARS et SARS fonctionnent comme des ligases E3 de NEDD8. De plus, RNF20 fonctionne également comme une NEDD8 E3 ligase responsable de la NEDDylation de l'histone H2B mais aussi de PARP1 - une des protéines acteurs clés dans la formation des SG
Understanding how organisms respond to environmental stress has critical implications both on quality of life and treatment of diseases. Organisms have developed a series of sophisticated processes to detect and repair such damages. A family of small proteins called the family of Ubiquitin molecules (Ubls), play a critical role in many aspects of the stress response. Defects in components of the Ubiquitin family are often found in pathologic conditions including cancer and neurodegenerative diseases. Understanding how the ubiquitin family is involved in the cellular stress response is an important step in the understanding of this process and can lead to the development of novel therapeutic approaches to treat diseases caused by malfunction of this system.One of the Ubls that has the highest identity and similarity to Ubiquitin is NEDD8. NEDD8 works in a similar manner to Ub, using a distinct conjugation machinery. NEDD8 modification is essential for maintaining the homeostasis of the cell as it plays a major role in the regulation of viability, growth, and development. Because of that, many components of NEDD8 have been found deregulated in many cancers. NEDD8 can modify a wide range of substrate proteins, including itself, which results in the creation of polyNEDD8 chains. Recently the presence of polyNEDD8 chains has been linked to the regulation of cell death – apoptosis and parthanatos. Moreover, it has been recently reported that NEDD8 during proteotoxic stress can be employed by the Ub conjugation machinery. This results in the creation of hybrid NEDD8 chains where except for NEDD8, we can also find Ub and SUMO, as recent papers have shown that NEDD8 has the ability to modify Ub and SUMO-2. The presence of the hybrid NEDD8 chains was linked with the creation of nuclear aggregates formed during proteotoxic stress, which can play a potential protective role during stress exposure.Knowing how important the regulation of proteins through NEDDylation is, we were also aware of the lack of knowledge about the machinery that plays a role in the creation and deconjugation of different NEDD8 entities. So far two deNEDDylating enzymes were reported but no enzyme was tested for its ability to recognise and process the hybrid NEDD8 chains. Moreover, our knowledge about E3 ligases that are responsible for substrate NEDDylation, even though expanding, is still very limited. Additionally, NEDD8 having ten lysines through which it can modify itself, can generate a very broad range of signals through polyNEDD8 and hybrid NEDD8 chain formation, which can be recognised similarly to polyUb chains, yet no studies have focused on determining their interactors so far.In this work, we focused on exploring the beforementioned unknown elements of the NEDD8 conjugation and deconjugation machineries. Using chemical biology approaches we tested a variety of enzymes and determined that polyNEDD8 chains are exclusively processed by the NEDP1 enzyme, however, deconjugation of hybrid NEDD8 chains requires the coordinated action of different deconjugating enzymes with distinct specificity for Ub or SUMO. We also employed chemically synthesized NEDD8-NEDD8 and NEDD8-Ub dimers in order to look for their interactors and used the gathered data to deepen our knowledge about the biology of hybrid NEDD8 chains in nuclear aggregates. Using NEDD8-Dha probes we identified a group of proteins that are potentially involved in the NEDDylation machinery. Through biological confirmation of obtained results we have shown that tRNA ligases – GARS and SARS are working as NEDD8 E3 ligases. Moreover, RNF20 is also working as a NEDD8 E3 ligase responsible for NEDDylation of histone H2B but also PARP1 – one of the proteins that are key players in the formation of SG

The BCA2 E3 ligase is expressed in a majority of invasive breast cancers. BCA2 has inherent autoubiquitination activity which contributes to cell migration and proliferation processes. Here, ten novel BCA2 binding proteins were found using yeast and bacterial screening. Two of which were human homolog of Rad23 variant A (hHR23a) and 14-3-3σ. In vivo and in vitro assays confirmed that both hHR23a and 14-3-3σ bound BCA2 and were co-expressed with BCA2 in breast cancer cells. Interaction of BCA2 with hHR23a and 14-3-3σ affect the autoubiquitination and auto-degradation activity of BCA2. Multi-ubiquitination of hHR23a-bound BCA2 was dramatically lower than that of free BCA2, this corresponded to increased BCA2 expression and half-life. Furthermore, phosphorylated BCA2 protein was stabilized by interaction with 14-3-3σ, via substrate inhibition of BCA2 autoubiquitination. High expression of BCA2 is correlated with grade in breast cancer and regulation of this E3 ligase’s activity may be important to cancer progression.

Parkinson’s disease (PD) is the most common neurodegenerative disorder after Alzheimer’s disease with approximately 7 million patients worldwide which are predicted to get doubled by 2030. The most common form of PD is the sporadic form with no known cause. Amongst many factors responsible for the pathogenesis of sporadic PD, Cell cycle reentry (CCE) with subsequent DNA synthesis in at-risk dopaminergic neurons has been recently identified as the cause of neuronal cell death. Neurons are post-mitotic cells which never divide, but in lieu of their physiological demands certain cell cycle proteins are utilized. However, under the influence of various stressors cell cycle is re-activated in a full blown manner and owing to mitotic incompetence of neurons, drive them to death. Mounting evidence has outlined the causal role of CCE in the pathogenesis of PD and other neurodegenerative disorders. Moreover, certain protective proteins such as ubiquitin E3 ligases and heat shock proteins play crucial role in maintaining protein homeostasis and in alleviating toxic protein burden in various neurodegenerative disorders thereby, promoting neuronal cell survival. The present work aims to study the involvement of cell cycle proteins in neuronal apoptosis and to underline the role of protective proteins especially HSP70 in neuronal cell viability. The study uses widely known toxin rotenone to mimic PD in SH-SY5Y neuroblastoma cell lines. The results show upregulation of cyclin E with subsequent attenuation of ubiquitin E3 ligase parkin and HSP70 in response to rotenone administration. Further, screening of HSP70 inducing biomolecules have clearly outlined their neuroprotective potential in attenuating CCE led neuronal death and in modifying and reversing rotenone induced toxicity. Thus, the present work opens up a new avenue of using HSP70 inducing compounds to target CCE mediated neuronal death in PD which can be extended to other neurodegenerative disorders

碩士
國立臺灣大學
生化科技學系
106
EBV, also known as Human Herpesvirus 4 (HHV-4), is a member of Herpesviridae. EBV infects epithelial cells and B cells, since its association with several cancers, it is known as an oncovirus. To protect themselves from infection by viruses, host cells usually degrade viral proteins to inhibit viral replication. Earlier research showed that TRIM5α, an E3 ubiquitin ligase, is important to EBV virion assembly. Also, it has been found that immediate-early protein Rta and late protein BORF1, both important in replication of EBV, are ubiquitinated by TRIM5α. Ubiquitination of Rta and BORF1 affect EBV DNA replication and viral capsid assembly, which decrease efficiency of virus production. In a previous study, BSLF1 protein of EBV was found to have deubiquitinase activity although it was widely known as a primase. BSLF1 is able to deubiquitinate Rta and BORF1, suggesting that BSLF1 plays a role in defending host modification. The aim of this study is to elucidate the antagonism between BSLF1 and E3 ubiquitin ligase TRIM5α. First, GST pull-down assay and immunofluorescence analysis revealed that BSLF1 interacts directly with TRIM5α. To examine the deubiquitination of Rta and BORF1 by BSLF1, I used denature immunoprecipitation and demonstrated that BSLF1 deubiquitinates Rta and BORF1 that are ubiquitinated by TRIM5α. Moreover, this study found that overexpressing BSLF1 decreased the transcriptional activity of Rta in a transient transfection assay. By using a HEK293T cell clone that expresses BORF1, this study found that overexpressing of BSLF1 increases the half-life of BORF1, but overexpression of BSLF1 did not rescue the levels of BORF1 in cells. This study also showed that deubiquitination of Rta or BORF1 does not influence the transactivation activity or increase the stability. Therefore, I further investigated which types of poly-ubiquitin chain did BSLF1 counteract against TRIM5α. Through expressing of K63-only or K48-only types of HA-Ub, this study found that although TRIM5α added both K48 and K63 poly-ubiquitin chains to BORF1, BSLF1 mainly deubiquitinated K63 poly-ubiquitin chain. Together, this study reveals BSLF1 deubiquitination activity and the antagonistic relationship between BSLF1 and TRIM5α. Although more research on how different types of poly-ubiquitin chain affect EBV life cycle is needed, the counteraction between EBV viral protein and host’s post-translational modification is likely important for EBV to escape from inhibition of host cells.