18 research outputs found
Analyse du trafic et de la distribution intracellulaire de la protéine Gag du VIH-1 dans les cellules HEK 293T : importance de l'efficacité de la relùche virale
Mémoire numérisé par la Direction des bibliothÚques de l'Université de Montréal
Importance of the HSP90 molecular chaperoning pathway for antibody diversification by determining AID stability
La protĂ©ine AID (dĂ©aminase induite par lâactivation) joue un rĂŽle central dans la
réponse immunitaire adaptative. En désaminant des désoxycytidines en désoxyuridines au
niveau des gĂšnes immunoglobulines, elle initie lâhypermutation somatique (SHM), la
conversion génique (iGC) et la commutation isotypique (CSR). Elle est essentielle à une
rĂ©ponse humorale efficace en contribuant Ă la maturation de lâaffinitĂ© des anticorps et au
changement de classe isotypique. Cependant, son activitĂ© mutagĂ©nique peut ĂȘtre oncogĂ©nique 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
gĂ©nĂ©rer une rĂ©ponse immunitaire efficace tout en minimisant les risques de cancer et dâautoimmunitĂ©.
Un Ă©lĂ©ment de rĂ©gulation est le fait quâAID transite du cytoplasme vers le noyau
mais reste majoritairement cytoplasmique Ă lâĂ©quilibre. AID est par ailleurs plus stable dans le
cytoplasme que dans le noyau, ce qui contribue Ă rĂ©duire sa prĂ©sence Ă proximitĂ© de lâADN.
Le but de cette thĂšse Ă©tait dâidentifier de nouveaux partenaires et dĂ©terminants dâAID
régulant sa stabilité et ses fonctions biologiques. Dans un premier temps, nous avons identifié
AID comme une nouvelle protĂ©ine cliente dâHSP90. Nous avons montrĂ© quâHSP90 interagit
avec AID dans le cytoplasme, ce qui empĂȘche la poly-ubiquitination dâAID et sa dĂ©gradation
par le protĂ©asome. En consĂ©quence, lâinhibition dâHSP90 rĂ©sulte en une diminution
significative des niveaux endogĂšnes dâAID et corrĂšle avec une rĂ©duction proportionnelle de ses
fonctions biologiques dans la diversification des anticorps mais aussi dans lâintroduction de
mutations aberrantes. Dans un second temps, nous avons montrĂ© que lâĂ©tape initiale dans la
stabilisation dâAID par la voie de chaperonnage dâHSP90 dĂ©pend dâHSP40 et dâHSP70. En
particulier, la protéine DnaJa1, qui fait partie de la famille des protéines HSP40s, limite la
stabilisation dâAID dans le cytoplasme. La farnĂ©sylation de DnaJa1 est importante pour
lâinteraction entre DnaJa1 et AID et moduler les niveaux de DnaJa1 ou son Ă©tat de farnĂ©sylation
impacte Ă la fois les niveaux endogĂšnes dâAID mais aussi la diversification des anticorps. Les
souris DNAJA1-/- prĂ©sentent une rĂ©ponse immunitaire compromise en cas dâimmunisation, qui
est dĂ»e Ă des niveaux rĂ©duits dâAID et un dĂ©faut de commutation de classe. Dans un troisiĂšme
temps, nous avons montrĂ© que la protĂ©ine AID est intrinsĂšquement plus instable que sesprotĂ©ines paralogues APOBEC. Nous avons identifiĂ© lâacide aspartique en seconde position
dâAID ainsi quâun motif semblable au PEST comme des modulateurs de la stabilitĂ© dâAID. La
modification de ces motifs augmente la stabilitĂ© dâAID et rĂ©sulte en une diversification des
anticorps plus efficace.
En conclusion, lâinstabilitĂ© intrinsĂšque dâAID est un Ă©lĂ©ment de rĂ©gulation de la
diversification des anticorps. Cette instabilité est en partie compensée dans le cytoplasme par
lâaction protective de la voie de chaperonnage DnaJa1-HSP90. Par ailleurs, lâutilisation
dâinhibiteurs dâHSP90 ou de farnĂ©syltransfĂ©rases pourrait ĂȘtre un outil intĂ©ressant pour la
modulation indirecte des niveaux dâAID et le traitement de lymphomes/leucĂ©mies 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 AIDâs 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
Common clonal origin of chronic myelomonocytic leukemia and B-cell acute lymphoblastic leukemia in a patient with a germline CHEK2 variant
Hematological malignancies are broadly divided into myeloid and lymphoid neoplasms, reflecting the two major cellular lineages of the hematopoietic system. It is generally rare for hematological malignancies to spontaneously progress with a switch from myeloid to lymphoid lineage. We describe the exceptional case of a patient who sequentially developed myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), and B-cell acute lymphoblastic leukemia (B-ALL), as well as our investigation into the underlying pathogenesis. Using whole-exome sequencing (WES) performed on sorted CMML and B-ALL cell fractions, we identified both common and unique potential driver mutations, suggesting a branching clonal evolution giving rise to both diseases. Interestingly, we also identified a germline variant in the cancer susceptibility gene CHEK2 We validated that this variant (c.475T > C; p.Y159H), located in the forkhead-associated (FHA) domain, impairs its capacity to bind BRCA1 in cellulo. This unique case provides novel insight into the genetics of complex hematological diseases and highlights the possibility that such patients may carry inherited predispositions
A mechanism for the suppression of homologous recombination in G1 cells
DNA repair by homologous recombination (HR)(1) is highly suppressed in G1 cells(2,3) to ensure that mitotic recombination occurs solely between sister chromatids(4). Although many HR factors are cell cycle-regulated, the identity of the events that are both necessary and sufficient to suppress recombination in G1 cells is unknown. Here we report that the cell cycle controls the interaction of BRCA1 with PALB2-BRCA2 in order to constrain BRCA2 function to the S/G2 phases. We found that the BRCA1-interaction site on PALB2 is targeted by an E3 ubiquitin ligase composed of KEAP1, a PALB2-interacting protein(5), in complex with CUL3-RBX1(6). PALB2 ubiquitylation suppresses its interaction with BRCA1 and is counteracted by the deubiquitylase USP11, which is itself under cell cycle control. Restoration of the BRCA1-PALB2 interaction combined with the activation of DNA end resection is sufficient to induce HR in G1, as measured by RAD51 recruitment, unscheduled DNA synthesis and a CRISPR/Cas9-based gene targeting assay. We conclude that the mechanism prohibiting HR in G1 minimally consists of the suppression of DNA end resection coupled to a multi-step block to BRCA2 recruitment to DNA damage sites that involves the inhibition of BRCA1-PALB2-BRCA2 complex assembly. We speculate that the ability to induce HR in G1 cells with defined factors could spur the development of gene targeting applications in non-dividing cells
Current gene panels account for nearly all homologous recombination repair-associated multiple-case breast cancer families
It was hypothesized that variants in underexplored homologous recombination repair (HR) genes could explain unsolved multiple-case breast cancer (BC) families. We investigated HR deficiency (HRD)-associated mutational signatures and second hits in tumor DNA from familial BC cases. No candidates genes were associated with HRD in 38 probands previously tested negative with gene panels. We conclude it is unlikely that unknown HRD-associated genes explain a large fraction of unsolved familial BC
Regulation of activation-induced deaminase stability and antibody gene diversification by Hsp90
Hsp90 stabilizes and prevents degradation of cytoplasmic activation-induced deaminase
Active nuclear import and cytoplasmic retention of activation-induced deaminase
The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.This
work was supported by the Canadian Institutes of Health Research (MOP 84543)
and a Canada Research Chair (to J.M.D.). A.O. was supported by a fellowship
from the Canadian Institutes of Health Research Cancer Training Program at the
IRCM. V.A.C. was supported in part by a Michel Saucier fellowship from the
Louis-Pasteur Canadian Fund through the University of Montreal