Heat shock proteins (Hsps) are a class of molecular chaperones which were first discovered as proteins up-regulated in response to heat stress in Drosophila. Later, it was found that these set of proteins get up-regulated as a general stress response associated with destabilization of native protein structures. Over a period of time, intricate involvement of Hsps in various biological processes has been well established. Heat shock protein 90 (Hsp90) is one of the important representative of this class of proteins. Hsp90 is an essential molecular chaperone which is evolutionarily conserved. It has a selective set of proteins to chaperone called as clients, which majorly include transcription factors and protein kinases. Through its interaction with its clients it modulates cell cycle, signal transduction, differentiation, development and evolution. Previous studies from Candida, Leishmania and Plasmodium have implicated Hsp90 to be involved in stage transition and growth. It is also critically involved in regulating growth of other protozoans such as Dictyostelium, Entamoeba and Trypanosoma. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. In Plasmodium falciparum, Hsp90 plays a critical role in stage transition. The parasite inside the human RBC develops from ring to trophozoite to schizont stage and inhibition of Hsp90 using specific pharmacological inhibitor arrests the growth of parasite at ring stage. In Dictyostelium, it has been observed that Hsp90 function is required for development. Inhibition of Hsp90 causes mound arrest and stops the cells from entering to its next developmental stage, fruiting bodies. In parallel, Hsp90 in Candida has been shown to be involved in morphogenesis. In nature Candida exists as a single cell yeast form and upon entry into the human host these yeast forms undergo morphogenesis to form virulent filamentous fungi. Inhibition of Hsp90 mimics temperature mediated morphogenesis. All together, these studies suggest that Hsp90 functions in a context dependent manner and each biological system explored has given new insights into the Hsp90 biology. Giardia lamblia, a protozoan parasite of humans and animals, is an important cause of diarrheal disease causing significant morbidity and also mortality in tropical countries. In the present study we focus on the biology of Hsp90 from Giardia lamblia. Giardia has a biphasic life cycle with infective cyst stage and pathogenic trophozoite stage. These cysts are present in the environment and enter mammalian host through oral route. They undergo a process called as excystation in the intestine giving rise to trophozoites. The trophozoites so formed colonize the upper part of the small intestine which causes the symptoms of giardiasis. Some of the trophozoites escape from the nutrition rich milieu of the upper part of small intestine to the lower part. In this region, trophozoites undergo a process called as encystation, wherein each trophozoite forms a cyst which escapes through faeces back into the environment. As seen in the life cycle of Giardia there are two major biological transitions, excystation and encystation; and till date no definitive player or pathway is known to regulate these processes. With the knowledge of Hsp90 playing an important role in similar biological transitions in other organisms we were encouraged to study role of Hsp90 in Giardia lamblia. Trans-splicing based generation of a full length Hsp90 in Giardia lamblia To understand the role of Hsp90, we first carried out sequence alignment of Hsp90 predicted ORFs in Giardia genome with yeast Hsp90. On alignment we observed that Hsp90 in Giardia is discontinuous and is annotated to be encoded by two different ORFs. Hsp90 in most organisms is coded by a single ORF with none to many cis-spliced introns. In a relatively intron poor organism G. lamblia, cytosolic Hsp90 is coded by two different ORFs separated by 777 kb in the genome. On multiple sequence alignment, we noticed that these two ORFs correspond to two independent regions of the Hsp90 protein. The ORFs are designated as hspN and hspC, containing the N-terminal and the C-terminal region of the protein respectively. We began our study by sequencing whole genome of Giardia lamblia clinical strain. Our genome sequencing confirmed the split nature of hsp90 and showed high ‘synteny’ between the other sequenced isolates. Using PCR based approach we have ruled out the possibility of having a full length gene in the genome. In contradiction to the genome result, we have observed a higher molecular weight protein in the lysate on proteomic analysis which was further confirmed by western blotting. The protein was observed to have a molecular weight of 80 kDa which could be a resultant of combination of two ORFs, suggesting the presence of a full length mRNA for Hsp90. PCR amplification using primers against both the fragments resulted in amplification of 2.1 kb product from the RNA pool of Giardia. Sequencing of this product showed that hspN and hspC were stitched together to form a mature messenger for full length Hsp90. In total our results suggest a post transcriptional process, trans-splicing, to be involved in the construction of Hsp90. The transition marked by this fusion coincides with the canonical GU¬AG splice site transitions as observed in other eukaryotes. Interestingly, a 26 nt near-complementary region was observed inside and upstream of hspN and hspC ORFs respectively. Put together these results suggest that the 26 nt complementary region acts as the positioning element to bring these two precursors in spatial proximity. With efficient spliceosomal activity these two precursor forms are trans-spliced to generate a full length cytosolic Hsp90 in Giardia. There are only four genes which have cis-spliced introns in the Giardia genome and the core components of the spliceosomal machinery are also present. The presence of canonical splice site in both the transcripts suggests that these transcripts are fused together by the spliceosomal machinery by the phenomenon of trans-splicing. The formation of full length Hsp90 RNA by its fragmented gene is the first example of trans-splicing in Giardia. To understand, are there any other genes which are also similarly trans-spliced we have carried out shotgun proteomic analysis of the total cell lysate obtained from Giardia trophozoites. Using Hsp90 as template, in our proteomic datasets, we have designed an algorithm for identification of additional trans-spliced gene products at the protein level. We have identified a total of 476 proteins of which hypothetical proteins constitute the major class followed by metabolic enzymes. We have compared the theoretical molecular weights for the identified proteins with the experimentally determined mass. Any discrepancy in the molecular mass was further analyzed and we assigned a gene to be potentially trans-spliced based on three criteria: if they were encoded by two or more different ORFs (loci), absence of a single full length counterpart and presence of splice sites with branch point and positional elements. Using this algorithm we were able to identify dynein as a potential candidate of trans-splicing reaction which was confirmed by the nucleotide sequence analysis of the predicted ORFs. Interestingly, dynein gene fragments were observed to be scattered on different chromosomes with minor splice sites unlike hsp90 genes. In vivo Expression of Hsp90 sub-fragments, HspN and HspC In the mature Hsp90 mRNA formed upon trans-splicing, 33 additional codons are present right between hspN and hspC sequences and they were acquired from the upstream region of hspC ORF. The 33 codons encode for an important region of Hsp90 which harbours the conserved catalytic “Arg” residue; suggesting that the full length Giardia Hsp90 (GlHsp90) formed could be an active ATPase. To confirm the same we have carried out in vitro characterization of trans-spliced Hsp90. Towards this, we have cloned, expressed and purified His tag-GlHsp90. As a first step, highly purified protein was used to assess its efficiency in binding to it cognate ligand, ATP, and the known inhibitors. Our binding studies show that GlHsp90 binds to ATP with a dissociation constant of 628 M and to its inhibitors, GA and 17AAG with 1.5 μM and 17.5 μM respectively. The bound ATP will be subsequently cleaved by Hsp90 which is an essential step in the chaperone cycle. As determined in our ATPase assay we observed that GlHsp90 hydrolyzes bound ATP with the catalytic efficiency of 4.4 × 10-5μM-1.min-1which confirms that Hsp90 generated upon trans-splicing is an active ATPase. The uniqueness of the hsp90 gene arrangement in Giardia posed a new question. Do these gene fragments also get translated? Our results suggest that HspN and HspC are poly¬adenylated. In order to determine the levels of these transcripts we performed qRT-PCR using primers specific to HspN, HspC and GlHsp90. We have observed that, in comparison with HspN transcript level, HspC and GlHsp90 transcripts are 15 and 75 folds higher respectively. To check for the presence of translation products of these transcripts, we have re-analyzed our proteomic datasets wherein we could identify peptides corresponding to HspN and HspC in their respective molecular weight region, 45 to 35 kDa. To confirm the proteomic data, western blot analysis was performed for trophozoite lysate on both 1D and 2D gels using anti-HspN antibody. Two specific bands (1D) / spots (2D) corresponding to the full length Hsp90 and HspN were identified. Gel filtration analysis revealed that HspN co¬eluted with full length Hsp90 thereby suggesting that both the proteins are in a same complex. With the background that HspN and HspC are present at the protein level, we asked if these fragments in combination can hydrolyse ATP. We reconstituted recombinant HspN and HspC in equimolar amounts and scored for the hydrolysis of ATP. However, no Pi release was observed. To determine whether HspN and HspC could modulate Hsp90 function, ATPase activity was monitored in the presence of HspN or HspC, in vitro. It was observed that ATPase activity was inhibited by both the fragments thus suggesting that HspN and HspC negatively regulate Hsp90 ATPase activity. Role of Hsp90 in Giardia encystation Giardia has a biphasic life cycle with proliferative trophozoites and latent cyst stage. In Giardia, in vitro encystation was established nearly two decades back by modulating the medium conditions. However, the mechanism and triggers underlying this transition are not well characterized. To understand whether Hsp90 has any role in this transition, in vitro conversion of trophozoites to cysts was achieved. The cysts obtained showed all the characteristic features of mature Giardia cyst with cyst wall protein 1 (CWP1) on the cyst wall and four nuclei as determined by immunofluorescence analysis. Further, the levels of Hsp90 in trophozoites were compared with mature cysts at both transcript and protein levels and it was found that cysts show more than 50% reduction in the level of Hsp90 in comparison with normal trophozoites. In accordance, exogenous inhibition of Hsp90 using 17AAG promoted the formation of cysts in vitro by 60 folds in a dose dependent manner; however, the window period of Hsp90 function compromise plays an important role in this process. Higher numbers of cysts were obtained from the cells treated with inhibitors during pre-encystation condition but inhibition of Hsp90 during encystation did not affect the formation of cysts, suggesting that Hsp90 down-regulation plays an important role during commitment towards encystation. To further show that cyst formation is a specific response to Hsp90 inhibition we have carried out encystation in the presence of metranidazole and from heat shocked cells; however, in both the conditions we did not observe any significant change in cyst formation, thus confirming that Hsp90 plays an important role during encystation in Giardia lamblia. Summary In Conclusion, Our study throws light on a unique aspect of Hsp90 biology in Giardia Lamblia, wherein the formation of the full length protein is dependent on a unique trans splicing reaction of its gene components representing different domains. We have also shown that HsP90 fragments, HspN and HspC, are also expressed in Trophozoites. Our in vitro data suggests that these fragments possibly regulate the function of Hsp90. Furthermore, the full length of Hsp90 plays an important role in stage transition in Giardia wherein inhibition of Hsp90 induces encystations. The study has opened many new avenues for research. Understanding the exact role of HspN and HspC in vivo will provide better appreciation for the evolution of such a complex biogenesis of an essential protein.

Our understanding of protein lysine methyltransferases and their substrates remains limited despite their importance as regulators of the proteome. The SMYD (SET and MYND domain) methyltransferase family plays pivotal roles in various cellular processes, including transcriptional regulation and embryonic development. Among them, SMYD2 is associated with oesophageal squamous cell carcinoma, bladder cancer and leukemia as well as with embryonic development. Initially identified as a histone methyltransferase, SMYD2 was later reported to methylate p53, the retinoblastoma protein pRb and the estrogen receptor ERalpha and to regulate their activity. Our proteomic and biochemical analyses demonstrated that SMYD2 also methylates the molecular chaperone HSP90 on K209 and K615. We also showed that HSP90 methylation is regulated by HSP90 co-chaperones, pH, and the demethylase LSD1. Further methyltransferase assays demonstrated that SMYD2 methylates lysine K* in proteins which include the sequence [LFM]-₁-K*-[AFYMSHRK]+₁-[LYK]+₂. This motif allowed us to show that SMYD2 methylates the transcriptional co-repressor SIN3B, the RNA helicase DHX15 and the myogenic transcription factors SIX1 and SIX2. Finally, muscle cell models suggest that SMYD2 methyltransferase activity plays a role in preventing premature myogenic differentiation of proliferating myoblasts by repressing muscle-specific genes. Our work thus shows that SMYD2 methyltransferase activity targets a broad array of substrates in vitro and in situ and is regulated by intricate mechanisms.

Orientador: Carlos Henrique Inácio Ramos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T13:20:36Z (GMT). No. of bitstreams: 1 Murakami_LeticiaMariaZanphorlin_D.pdf: 5383539 bytes, checksum: 1a45d203e6e3c5a992791b8ce893aa36 (MD5) Previous issue date: 2014 / Resumo: Chaperonas moleculares e proteínas de choque térmico (Heat shock protein, Hsp) atuam contra a agregação e o enovelamento incorreto de proteínas, que são os agentes causais de doenças neurodegenerativas, como por exemplo, Alzheimer e Parkinson. A Hsp90 é uma das mais importantes chaperonas moleculares, considerada essencial para a viabilidade celular em eucariotos, pois está associada com a maturação de proteínas atuantes na sinalização e ciclo celular. Além disso, foi demonstrado que a Hsp90 está envolvida na estabilização do fenótipo tumoral de diversos tipos de câncer, destacando a sua importância biomédica. A interação com co-chaperonas, proteínas auxiliares das chaperonas, permite que a Hsp90 atue como uma proteína "hub", ou seja, um ponto central de regulação de diversas proteínas. Muitas dessas co-chaperonas possuem um ou mais domínios do tipo TPR (do inglês, tetratricopeptide repeat) que interagem com o C-terminal da Hsp90. No presente projeto de doutorado, investigamos as características estruturais e termodinâmicas da interação entre o domínio C-terminal da Hsp90 (C-Hsp90) e a co-chaperona TPR Tom70 humana, utilizando técnicas de reação-cruzada acoplada à espectrometria de massas (LC-MS/MS), calorimetria de titulação isotérmica (ITC), espalhamento de raios-X à baixos ângulos (SAXS) e modelagem molecular. Os resultados de LC-MS/MS e ITC evidenciaram novas regiões na interação do complexo C-Hsp90/Tom70 que envolve a hélice A7 presente na Tom70 e experimentos de SAXS revelaram a estrutura em baixa resolução das proteínas C-Hsp90, Tom70 e do complexo C-Hsp90/Tom70. Além disso, investigamos o efeito do celastrol, um composto com potencial atividade anti-câncer, na conformação e na função da Hsp90. Na presença do composto, a Hsp90 sofre um processo de oligomerização e a natureza dos oligômeros foi determinada por ferramentas bioquímicas e biofísicas, tais como espalhamento dinâmico de luz (DLS), cromatografia de exclusão molecular analítica acoplada a espalhamento de luz em multiângulos (SEC-MALS) e eletroforese em gel nativo. Interessantemente, a oligomerização induzida pelo celastrol não afetou a atividade de proteção da Hsp90 contra a agregação protéica e a capacidade de ligação as co-chaperonas com enovelamento tipo TPR. Este é o primeiro trabalho a apontar um possível mecanismo para a ação do celastrol sobre a Hsp90. Coletivamente, nossos resultados e descobertas contribuem para uma melhor compreensão dos mecanismos moleculares relacionados à interação entre chaperonas e co-chaperonas, bem como, chaperonas e potenciais ligantes. / Abstract: Molecular chaperones and heat shock proteins (Hsp) act against protein aggregation and misfolding, which are the causal agents of neurodegenerative diseases such as Alzheimer and Parkinson. Hsp90 is one of the most important molecular chaperones, considered essential for cell viability in eukaryotes, since it is associated with the maturation of proteins involved in cell cycle and signaling. In addition, it was demonstrated that Hsp90 is implicated in the stabilization of the tumor phenotype of various types of cancer, highlighting its biomedical importance. The interaction with co-chaperones, auxiliary proteins of chaperones, allows that Hsp90 acts as a hub, being a central point for regulation of several other proteins. Many of these co-chaperones have one or more TPR domains that interact with the C-terminus of Hsp90. In this PhD project, we investigated structural and thermodynamic characteristics of the interaction between the C-terminus domain of Hsp90 (C-Hsp90) and the TPR co-chaperone human Tom70, using techniques of cross-linking coupled with mass spectrometry (LC-MS/MS), isothermal titration calorimetry (ITC), small angle X-ray scattering (SAXS) and molecular modeling. The results of LC-MS/MS and ITC revealed new regions involved in the interaction of the C-Hsp90 with Tom70, which encompasses the A7 helix from Tom70, and SAXS experiments unveiled the low resolution structure of the proteins C-Hsp90, Tom70 and the C-Hsp90/Tom70 complex. In addition, we investigated the effect of celastrol, a compound with a potential anti-cancer activity, on the conformation and function of Hsp90. In the presence of celastrol, Hsp90 undergoes oligomerization and the nature of the oligomers was determined by biochemical and biophysical tools such as dynamic light scattering (DLS), size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and native gel electrophoresis. Interestingly, the celastrol-induced oligomerization did not affect the protective activities of Hsp90 against protein aggregation or the capacity to bind TPR co-chaperones. This is the first study to point out a possible mechanism for the action of celastrol on Hsp90. Collectively, our findings contribute to a better understanding of the molecular mechanisms associated to the interaction between chaperones and co-chaperones, as well as chaperones and potential ligands / Doutorado / Quimica Organica / Doutora em Ciências

Indiana University-Purdue University Indianapolis (IUPUI) / Heat shock protein (HSP) 90 is a molecular chaperone that regulates diverse cellular processes by facilitating activities of various protein clients. Recent studies have shown serum levels of the alpha cytoplasmic HSP90 isoform are elevated in newly diagnosed type I diabetic patients, thus distinguishing this protein as a potential biomarker for pre-clinical type I diabetes mellitus (TIDM). This phase of disease is known to be associated with various forms of beta cell stress, including endoplasmic reticulum stress, insulitis, and hyperglycemia. Therefore, to test the hypothesis that HSP90 is released by these cells in response to stress, human pancreatic beta cells were subjected to various forms of stress in vitro. Beta cells released HSP90 in response to stimulation with a combination of cytokines that included IL-1β, TNF-α, and IFN-γ, as well as an agonist of toll-like receptor 3. HSP90 release was not found to result from cellular increases in HSP90AA1 gene or HSP90 protein expression levels. Rather, cell stress and ensuing cytotoxicity mediated by c-Jun N-terminal kinase (JNK) appeared to play a role in HSP90 release. Beta cell HSP90 release was attenuated by pre-treatment with tauroursodeoxycholic acid (TUDCA), which has been shown previously to protect beta cells against JNK-mediated, cytokine-induced apoptosis. Experiments here confirmed TUDCA reduced beta cell JNK phosphorylation in response to cytokine stress. Furthermore pharmacological inhibition and siRNA-mediated knockdown of JNK in beta cells also attenuated HSP90 release in response to cytokine stress. Pharmacological inhibition of HSP90 chaperone function exacerbated islet cell stress during the development of TIDM in vivo; however, it did not affect the overall incidence of disease. Together, these data suggest extracellular HSP90 could serve as a biomarker for preclinical TIDM. This knowledge may be clinically relevant in optimizing treatments aimed at restoring beta cell mass. The goal of such treatments would be to halt the progression of at-risk patients to insulin dependence and lifelong TIDM.

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<p>Human actions and the resultant global warming are leading to considerable environmental changes that are negatively impacting marine ecosystems and their biodiversity. Luidia clathrata, a starfish species, is essential to the marine ecosystem, and understanding its sensitivity to stressors can help predict its future adaptations and role in the reef ecosystem. The study involved subjecting L. clathrata to thermal stress by incrementally raising the temperature by 1°C each day for a period of seven days. Physiological responses were evaluated on two separate occasions: day 1, which corresponded to the acute stress response, and day 7, which corresponded to the chronic stress response. The results showed a minor increase in phagocytic activity during acute thermal stress, but a significant decrease during chronic exposure. Although there was a slight decrease in total coelomic plasma protein during acute thermal stress, it significantly increased during post-chronic exposure. The amputated starfish avoided using the injured arm when righting themselves, indicating the development of neurosensory potential. Total cell count increased slightly in all stressed groups during acute stress but decreased after prolonged exposure to stressors. The mortality rate of the temperature-stressed groups was 33%, indicating that prolonged exposure to temperatures exceeding expected future temperatures could be harmful to L. clathrata. To support the hypothesis at the molecular level, RNA/DNA ratios and Heat shock protein gene 90, a molecular marker for cellular stress, were studied. Although no significant differences were observed in transcriptomic level, the temperature-stressed group showed slightly upregulated hsp90 gene expression. The findings indicate that L. clathrata responds to stress similarly to vertebrates, highlighting the potential impact of climate change on marine ecosystems. This study provides a baseline for comprehending the stress response of starfish, and further research is recommended with a larger sample size and over a more extended period. It is interesting to note that the gonad and body wall extracts of starfish exhibit significant inhibitory activity against various tested pathogens. The findings suggest that starfish extracts may have potential medicinal uses as antimicrobial agents. However further research is needed to understand the mechanisms of action behind these inhibitory activities and to identify the specific compounds responsible for them.</p>

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La protéine AID (déaminase induite par l’activation) joue un rôle central dans la réponse immunitaire adaptative. En désaminant des désoxycytidines en désoxyuridines au niveau des gènes immunoglobulines, elle initie l’hypermutation somatique (SHM), la conversion génique (iGC) et la commutation isotypique (CSR). Elle est essentielle à une réponse humorale efficace en contribuant à la maturation de l’affinité des anticorps et au changement de classe isotypique. Cependant, son activité mutagénique peut être oncogénique et causer une instabilité génomique propice au développement de cancers et de maladies autoimmunes. Il est donc critique de réguler AID, en particulier ses niveaux protéiques, pour générer une réponse immunitaire efficace tout en minimisant les risques de cancer et d’autoimmunité. Un élément de régulation est le fait qu’AID transite du cytoplasme vers le noyau mais reste majoritairement cytoplasmique à l’équilibre. AID est par ailleurs plus stable dans le cytoplasme que dans le noyau, ce qui contribue à réduire sa présence à proximité de l’ADN. Le but de cette thèse était d’identifier de nouveaux partenaires et déterminants d’AID régulant sa stabilité et ses fonctions biologiques. Dans un premier temps, nous avons identifié AID comme une nouvelle protéine cliente d’HSP90. Nous avons montré qu’HSP90 interagit avec AID dans le cytoplasme, ce qui empêche la poly-ubiquitination d’AID et sa dégradation par le protéasome. En conséquence, l’inhibition d’HSP90 résulte en une diminution significative des niveaux endogènes d’AID et corrèle avec une réduction proportionnelle de ses fonctions biologiques dans la diversification des anticorps mais aussi dans l’introduction de mutations aberrantes. Dans un second temps, nous avons montré que l’étape initiale dans la stabilisation d’AID par la voie de chaperonnage d’HSP90 dépend d’HSP40 et d’HSP70. En particulier, la protéine DnaJa1, qui fait partie de la famille des protéines HSP40s, limite la stabilisation d’AID dans le cytoplasme. La farnésylation de DnaJa1 est importante pour l’interaction entre DnaJa1 et AID et moduler les niveaux de DnaJa1 ou son état de farnésylation impacte à la fois les niveaux endogènes d’AID mais aussi la diversification des anticorps. Les souris DNAJA1-/- présentent une réponse immunitaire compromise en cas d’immunisation, qui est dûe à des niveaux réduits d’AID et un défaut de commutation de classe. Dans un troisième temps, nous avons montré que la protéine AID est intrinsèquement plus instable que sesprotéines paralogues APOBEC. Nous avons identifié l’acide aspartique en seconde position d’AID ainsi qu’un motif semblable au PEST comme des modulateurs de la stabilité d’AID. La modification de ces motifs augmente la stabilité d’AID et résulte en une diversification des anticorps plus efficace. En conclusion, l’instabilité intrinsèque d’AID est un élément de régulation de la diversification des anticorps. Cette instabilité est en partie compensée dans le cytoplasme par l’action protective de la voie de chaperonnage DnaJa1-HSP90. Par ailleurs, l’utilisation d’inhibiteurs d’HSP90 ou de farnésyltransférases pourrait être un outil intéressant pour la modulation indirecte des niveaux d’AID et le traitement de lymphomes/leucémies et de maladies auto-immunes causés par AID. / Activation induced deaminase (AID) plays a central role in adaptive immunity. AID deaminates deoxycytidine to deoxyuridine in defined regions of the immunoglobulin (Ig) genes and initiates somatic hypermutation (SHM), gene conversion (iGC) and class switch recombination (CSR). While being essential for an effective immune response by underpinning antibody affinity maturation and isotype switching, the mutagenic activity of AID can also be oncogenic and causes genomic instability leading to the development of cancer, or exacerbate autoimmune diseases. Therefore, AID regulation, including the control of its protein level, is central to balancing effective immunity with cancer/autoimmunity. Notably, AID shuttles between the cytoplasm and the nucleus but is predominantly cytoplasmic at steady-state, with cytoplasmic AID being much more stable than nuclear AID. These regulatory steps contribute to limit the exposure of the genome to AID but their mechanisms are unknown. This thesis aimed at identifying AID partners and intrinsic determinants regulating its stability and modulating its biological functions. Firstly, we identified AID as a novel HSP90 client protein. We demonstrated that HSP90 interacts with AID in the cytoplasm and prevents its polyubiquitination and subsequent proteasomal degradation. Consequently, HSP90 inhibition results in a significant reduction of endogenous AID levels and correlates with a proportional reduction in both AID-mediated antibody diversification and off-target mutations. Secondly, we showed that the first step in the HSP90 molecular chaperoning pathway and stabilization is the interaction of AID with the HSP40 and HSP70 system. In fact, a specific HSP40 protein, DnaJa1, is the limiting step in cytoplasmic AID stabilization. DnaJa1 farnesylation is required for DnaJa1-AID interaction and modulation of DnaJa1 levels or its farnesylation impacts endogenous AID levels and antibody diversification. In vivo, DnaJa1- deficient mice display compromized response to immunization, resulting from reduced AID protein levels and isotype switching. Thirdly, we found that AID is intrinsically less stable than its APOBEC paralogs. We identified the AID N-terminal aspartic acid residue at position two and an internal PEST-like motif as destabilizing modulators of AID protein turnover. Disruption of these motifs increases AID protein stability and antibody diversification.We conclude that AID’s intrinsic instability directly contributes to regulating antibody diversification. This intrinsic instability is at least partially compensated for in the cytoplasm by the protective action of the DnaJa1-HSP90 molecular chaperoning pathway. Pharmacologically targeting AID in an indirect way, by using HSP90 or farnesyltransferase inhibitors, could be relevant for treating some AID-associated lymphomas/leukemias and/or autoimmune diseases.

No description available.