Heat shock proteins function as molecular chaperones which help protein folding and prevent protein aggregation. My study shows that celastrol, a pharmacological compound capable of up-regulating the levels of heat shock proteins, inhibits cell death and protein aggregation caused by expanded polyglutamine containing protein, and the protective effects of celastrol are dependent on heat shock factor 1. These results suggest the potential of celastrol as a therapeutic agent in the treatment of polyglutamine diseases. Sumoylation is a protein modification which plays diverse roles in regulating the target proteins. My study shows that lamin A is a target of protein sumoylation, and two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. My results also indicate that sumoylation is important for the normal localization of lamin A, and support a role for altered sumoylation in the underlying molecular mechanism of cardiomyopathies associated with the E203G/E203K lamin A mutations. In the third project, my results show that amyloid precursor protein is another target of SUMO modification, and sumoylation of amyloid precursor protein reduces the levels of amyloid β aggregates, which are the primary causative factor for Alzheimer’s disease. My results provide a new mechanism for the generation of amyloid β, and indicate the potential of up-regulating activity of the cellular sumoylation machinery as an approach against Alzheimer’s disease. My results also provide the first demonstration that SUMO E2 enzyme exists in the lumen of the endoplasmic reticulum, extending the sub-cellular reach of sumoylation to include the regulation of proteins in secretory pathways.

No description available.

L'utilisation des antirétroviraux (ART) a permis une augmentation de la durée des patients infectés par le VIH. Par ailleurs, les comorbidités, retrouvées au cours du vieillissement physiologique, semblent être plus fréquentes et d'apparition plus précoce ce qui pourrait suggérer une modification du programme de vieillissement chez ces patients. L'étude ANRS EP45 « Aging » (clinicalTrials.gov, NCT01038999) a pour objectif d'analyser chez des patients infectés par le VIH traités ou non les mécanismes cellulaires connus pour être impliqués dans le vieillissement. Les PBMC d'une cohorte de 130 patients infectés par le VIH 1 appariés en âge et en sexe avec 49 sujets séronégatifs ont été analysés. Trois centres spécialisés (Marseille, Montpellier, Nice) ont recruté des patients infectés naïfs ou sous première ligne de traitement. Les résultats présentés dans ce manuscrit rapportent l'analyse des mitochondries et des lamines nucléaires. La maturation de la lamine A ne semble pas modifiée dans les PBMC de patients sous traitement contenant un inhibiteur de protéase. Cependant, ces cellules pourraient ne pas être le modèle le plus adapté pour explorer ce volet. D'autre part, l'infection est responsable d'anomalies mitochondriales dans les lymphocytes, partiellement corrigées par les traitements antirétroviraux qui modifient les mitochondries des monocytes moins sensibles à l'infection. Bien que les secondes générations de ART soient moins toxiques que les premières, leurs effets secondaires pourraient néanmoins, sur « le long terme » et/ou généralisés à l'ensemble de l'organisme, être l'un des facteurs modifiant le programme de vieillissement de ces patients. / Antiretroviral therapy (ART) has increased life expectancy in HIV-infected patients. Moreover, some age-related disorders were found to be more frequent in HIV infected and treated patients than in an age-matched general population, suggesting a modified time course of aging in HIV infected patients. The ANRS EP45 « Aging » study (clinicalTrials.gov, NCT01038999) investigated in PBMC from HIV-1 infected patients under treatment or not the cellular mechanisms known to be involved in aging. The study was performed on a cohort of 130 patients HIV-1 infected age- and sex-matched with 49 seronegative control subjects. Patients never treated with ART (naïve) or under first line were recruited by 3 AIDS centres (Marseille, Montpellier, Nice). Results presented here describe explorations of mitochondria and nuclear lamin. No alteration of lamin A maturation was detected in PBMC from HIV-1 infected patients under treatment with protease inhibitor. However, these cells could not be the most appropriate models to investigate lamin A-related aging pathway. On another hand, mitochondrial modifications were observed in lymphocytes from HIV infected naive patients. These alterations were only partly rescued by ART whereas its induced slight changes in monocytes that appeared to be less sensitive to infection. While second generation of ART are less toxic than the first one, their secondary effects, due to long term exposure and/or generalised to different tissues, could lead to a modified time course of aging in HIV infected patients.

As the so-called “Mothership of the Human Genome,” the cell nucleus must keep all vital genetic information safe, but accessible, inside a strong protective envelope. The inner membrane of the nuclear envelope is lined by tough but adaptable proteins called lamins. While lamins polymerize into fibrous structures that hold up the “walls” of the nucleus, they also serve as an internal scaffold for the complex machinery involved in DNA replication and gene expression. It is in this later role that we have been looking for clues to premature and possibly to normal aging. One type of lamins, Lamin A is made through an unusual pathway involving a lipid dependent cleavage of a larger precursor called prelamin A. The functional significance of this processing pathway is that prelamin A cannot assemble and is inhibitory of proper lamina formation. Pathological cases of immature lamin A accumulation include Hutchinson-Gilford progeria syndrome (HGPS) or Progeria characterized by premature aging and Restrictive Dermopathy (RD), a lethal prenatal disease. We have previously shown that accumulation of prelamin A leads to cell cycle arrest and drastic changes in expression of genes involved in cell cycle control, among those, several members of the FoxO family of transcription factors. The goal of this study was to determine the mechanisms by which accumulation of uncleavable prelamin A activates FoxO-mediated cell cycle arrest. Cells expressing an uncleavable form of Lamin A in an inducible manner were used to determine subcellular distribution of FoxO3a upon accumulation of prelamin A. This was done by indirect immunofluorescence and Western blotting. The proliferation rate of these cells and controls expressing wild type Lamin A was also determined by measuring the incorporation of BrdU into DNA. During these experiments, it was hypothesized and observed that overexpression of prelamin A leads to redistribution of FoxO3a from the cytoplasm of the cell to the nucleoplasm. Expression of FoxO3a target genes was accordingly increased, leading to a decrease in cell proliferation. The information obtained from this study could not only be of interest in broadening our knowledge of the mechanisms of quiescence and aging in general, but also could inform the discussion of the use of several therapeutics for the treatment of Progeria and other diseases that result from the accumulation of prelamin A.

There is a large variety of diseases caused by single-gene mutations. Although most of these conditions are rare, together they impose a significant burden to the population. This thesis describes clinical and genetic studies of two single-gene diseases: 1) Adult-onset autosomal dominant leukodystrophy with autonomic symptoms (ADLD) caused by LMNB1 gene duplications, and characterized by autonomic, pyramidal and cerebellar symptoms. Spinal cords of patients with ADLD were studied by MRI and found to be thin, with high signal intensity in white matter. Histopathology showed loss of myelinated fibres with some reactive gliosis. DNA samples from four different families with ADLD were obtained, and the LMNB1 gene was screened for duplications. Single nucleotide polymorphism array revealed LMNB1 duplications in all ADLD families. LMNB1 mRNA and protein levels were assessed in white blood cells using quantitative polymerase chain reaction and Western blot, and increased levels of LMNB1 mRNA and lamin B1 protein could be demonstrated. We concluded that spinal cord atrophy in patients with ADLD is a valuable differential diagnostic sign, and that increased levels of LMNB1 can be detected in peripheral blood. 2) Rippling muscle disease (RMD) is caused by CAV3 gene mutations. Clinical features are percussion-induced muscle mounding, –rapid contractions and undulating muscle contractions (rippling). The CAV3 gene was sequenced in 38 members of a family with RMD. Twenty-two individuals had clinical features of RMD. No muscle weakness was seen. All patients with signs of RMD carried the p.A46T CAV3 mutation, showing that the p.A46T mutation was benign and that the diagnosis can be made clinically. In vitro contracture test results from 10 of the subjects were collected, but no association between pathological test results and RMD was found.

Thesis (Ph. D.)--University of Washington, 2007. / Includes bibliographical references (leaves 79-99).

La dystrophie musculaire d'Emery-Dreifuss (EDMD) est caractérisée par des retractions précoces, une faiblesse et atrophie musculaire lentement progressive, et une atteinte cardiaque. Les mutations des gènes EMD et LMNA sont respectivement responsables de formes liées à l'X et de formes autosomiques de l'EDMD. Ces deux gènes codent pour des protéines de l'enveloppe nucléaire, l'émerine et les lamines A/C. Les mutations du gène FHL1 ont été impliquées dans d'autres cas d'EDMD liée à l'X. FHL1 codent pour FHL1A, FHL1B et FHL1C, protéines jamais décrites comme localisées à l'enveloppe nucléaire. Nous avons cherché à enrichir les connaissances sur la distribution subcellulaire des différentes isoformes de FHL1 dans les muscles squelettiques humains sains et malades. Nous avons mis en évidence que les isoformes FHL1 présentent à la fois une localisation cytoplasmique et nucléaire dans les myoblastes humains. Au noyau, FHL1B est fortement accumulé au niveau de l'enveloppe nucléaire où il interagit avec les lamines A/C et l'émerine. Cette localisation à l'enveloppe nucléaire est indépendante de l'expression de l'émerine ou des lamines A/C. La différenciation des myoblastes entraîne une forte réduction de l'expression de FHL1B et de son exclusion progressive du noyau, n'impliquant pas la protéine CRM-1. Nous avons mis en évidence l'augmentation de l'expression de FHL1B dans les myoblastes de deux patients atteints d'EDMD, l'un porteur d'une mutation dans le gène LMNA, l'autre dans le gène FHL1. En conclusion, la localisation spécifique de FHL1B et sa modulation dans les myoblastes de patients confirment les cas d'EDMD liés à FHL1 comme des pathologies de l'enveloppe nucléaire. / Emery-Dreifuss muscular dystrophy (EDMD) is characterized by the triad of early contractures, slowly progressive muscle wasting and weakness, and cardiac disease. Mutations in EMD and LMNA, encoding for the nuclear envelope (NE) proteins emerin and lamin A/C, are associated with X-linked and autosomal form of EDMD, respectively. The discovery that FHL, encoding FHL1A, FHL1B and FHL1C, is implicated in the pathogenesis of EDMD, raises the question of how a non-NE protein can be linked to emerin and lamin A/C. We aimed to provide knowledge of the subcellular distribution and expression of the various FHL1 isoforms in healthy and diseased human skeletal muscle. We found that FHL1 isoforms display a dual cytoplasmic and nuclear localization in human myoblasts. In addition, FHL1B strongly accumulated at the NE where it interacted with both lamin A/C and emerin. NE localization of FHL1B was independent of emerin and lamin A/C expression. Myoblast differentiation resulted in greatly reduced FHL1B protein expression and in the progressive nuclear exclusion of FHL1 protein isoforms. We have shown that chromosome region maintenance 1 (CRM1)-mediated nuclear export was not involved in the progressive decrease of nucleoplasmic FHL1B. Finally, we detected increased FHL1B protein levels in myoblasts of two patients with LMNA- and FHL1-related EDMD. Altogether, the specific localization of FHL1B and its modulation in disease-patient’s myoblasts confirmed FHL1-related EDMD as a NE disease.

La maladie de Charcot-Marie-Tooth (CMT) est une maladie neurologique héréditaire du système nerveux périphérique. A ce jour, près de 80 gènes sont décrits comme étant à l’origine d’une forme de CMT dont tous les modes de transmission sont connus. AR-CMT2A est due à une mutation faux-sens homozygote, c.892C>T, dans l’exon 5 du gène LMNA et conduit à la substitution d’une Arginine par une Cystéine (p.Arg298Cys) au sein d’un motif conservé du domaine central coil des Lamines de type A. L’étude présentée ici fait suite à un certain nombre d’observations ayant démontré la diminution de l’expression du gène dans les cellules de patients, et la perte d’interaction entre les Lamines A/C mutées et le facteur de transcription c-Jun. Or celui-ci participe au complexe régulateur AP-1 pour lequel le promoteur du gène LMNA possède deux éléments de fixation. L’ensemble du travail exposé dans ce manuscrit s’est donc basé sur l’hypothèse selon laquelle les Lamines de type A auraient la capacité de réguler leur propre expression et seraient capables, dans le nerf périphérique, d’établir des interactions avec des partenaires spécifiques du nerf périphérique. / Charcot-Marie-Tooth (CMT) disorders constitute a complex and heterogeneous group of hereditary motor and sensory neuropathies characterized by muscle weakness and wasting, foot and hand deformities and electrophysiological changes. Genetically, CMT is characterized by a great heterogeneity, with all modes of inheritance and more than 50 genes described to date. My PhD work focuses on AR-CMT2A, a rare autosomal recessive axonal form of CMT, due to a unique homozygous missense mutation c.892C>T in LMNA exon 5, which leads to the substitution of an arginine by a cysteine (p.Arg298Cys) within a conserved motif in the central rod domain of A-type Lamins. My work aimed at providing clues toward a better understanding of the physiopathological mechanisms underlying AR-CMT2A and is based on previous results for my research team, showing a decrease in the expression of LMNA in patients’ cells, and a loss of interaction between A-type Lamins and the transcription factor c-Jun in patients’ cells. c-Jun is a member of the AP-1 complex, a well-known dimeric transcription factor, and for which interestingly, the LMNA promoter has two binding sites. All the work outlined in this manuscript is based on the hypothesis that A-type Lamins, have the capacity to regulate their own expression and therefore, are also most probably involved in interactions with partners involved in gene regulation, in particular in the Peripheral Nerve System.

Les lamines sont le principal composant du nucléosquelette. Elles sont principalement localisées à l’enveloppe nucléaire, où elles interagissent avec la membrane nucléaire interne, les protéines associées à la chromatine ainsi qu’avec des modulateurs de la signalisation cellulaire. Le gène LMNA code pour la prélamine A et la lamine C. La région C-terminale de la prélamine A est prédite pour être désordonnée et est la cible de plusieurs événements de maturation. En effet, la protéine est farnésylée, coupée, carboxyméthylée, puis coupée à nouveau ; perdant finalement son groupement farnésyl. Un mutant de cette protéine, dont 50 acides aminés sont manquants, est responsable du syndrome d’Huchtinson-Gilford, appelé progéria (Eriksson et al., 2003). Chez ce mutant, appelé progérine, le site de coupure finale est absent et la protéine reste constitutivement farnésylée. La lamine A est connue pour interagir avec la protéine de la membrane nucléaire interne, l’émerine. L’absence d’émerine est responsable de la dystrophie musculaire d’Emery Dreifuss. L’émerine contient un LEM, suivi d’une région prédite comme désordonnée, essentielle pour l’auto-assemblage de l’émerine (Berk et al., 2014). L’oligomérisation de l’émerine régule ses interactions avec plusieurs partenaires à la membrane nucléaire interne et à la chromatine. Nous avions auparavant démontré que la région nucléoplasmique de l’émerine peut s’auto-associer pour former des filaments in vitro (Herrada et al., 2015) et nous avons récemment révélé que ces filaments sont capables d’interagir directement avec la queue de la lamine A (Samson et al., 2018). Ici, je me suis intéressé à l’analyse structurale des régions prédites comme désordonnées chez (1) l’émerine (2) la prélamine A. Dans le cas de l’émerine, j’ai analysé la conformation de la région nucléoplasmique d’émerine avant et après auto-assemblage, en travaillant avec l’émerine sauvage et plusieurs mutants entraînant des myopathies. J’ai montré que deux fragments de l’émerine 1-187 et 67-221 peuvent polymériser, tandis que leur région commune 67-187, reste toujours monomérique dans nos conditions (Samson et al., 2018). Nous avons aussi montré que le domaine LEM est au moins partiellement déplié au cours de l’assemblage de la région 1-187. J’ai également attribué les signaux RMN de la région désordonnée 67-170, dans le but d’étudier par la suite l’impact des phosphorylations de cette région sur la structure de l’émerine et sur ses propriétés d’auto-assemblage (Samson et al., 2016). Dans le cas de la lamine A, j’ai étudié la région C-terminale de la prélamine A, prédite comme dépliée et qui est le siège de nombreuses modifications post-traductionnelles. J’ai attribué les signaux RMN du peptide prélamine A ainsi que de son mutant progérine (Celli et al., 2018). J’ai montré que ces deux peptides sont en effet déplés et possèdent une hélice  transitoire très conservée. Je propose cette hélice comme site de liaison pour un partenaire encore non identifié. J’ai également démontré que le peptide prélamine A possède une tendance à s’auto-assembler. Cependant, la prélamine A et le peptide progérine sauvages et farnésylés, n’interagissent pas avec le domaine IgFold de la lamine A ni avec BAF, deux domaines associés avec la progéria. J’ai étudié par la suite les interactions de ces peptides avec deux autres partenaires associés à la progéria : la protéine de la membrane nucléaire interne SUN1 et la protéine associée à la chromatine RBBP4. SUN1 est également intrinsèquement désordonnée et très peu soluble dans nos conditions. Les résultats montrent que le peptide prélamine A ne lie pas RBBP4 mais pourrait avoir besoin de la partie C-terminale qui la précède. Cependant, RBBP4 lie directement le partenaire de la lamine BAF. Sur les bases de ces résultats, je propose une série d’expériences pour identifier les détails moléculaires des interactions entre la queue C-terminale de la lamine A, BAF et RBBP4. / Lamins are the main components of the nucleoskeleton. They are primarily located at the nuclear envelope, where they interact with inner nuclear membrane proteins, chromatin-associated proteins and cell signaling modulators. The LMNA gene codes for prelamin A and lamin C. The C-terminal region of prelamin A is predicted to be unfolded and is the target of several maturation events. Indeed, the protein is farnesylated, cleaved, carboxymethylated and cleaved again; losing eventually its farnesyl group. A mutant of this protein, lacking 50 amino acids, is responsible for the Hutchinson-Gilford Progeria Syndrome (Eriksson et al., Nature 2003). In this mutant, called progerin, the final cleavage site is absent and the protein stays constitutively farnesylated. Lamin A is reported to interact with the inner nuclear membrane protein emerin. Lack of emerin is responsible for Emery Dreifuss Muscular Dystrophy. Emerin contains a folded LEM domain, followed by a region that is predicted to be disordered and is essential for emerin self-assembly (Berk et al., 2014). Emerin oligomerization regulates its interaction with several partners at the inner nuclear membrane and at the chromatin. We previously showed that the nucleoplasmic region of emerin can self-assemble to form curvilinear filaments in vitro (Herrada et al., 2015) and we recently revealed that these filaments are able to directly bind to the lamin A tail (Samson et al., 2018).Here I focused on the structural analysis of regions that are predicted to be unfolded in (1) emerin, (2) prelamin A. In the case of emerin, I analysed the conformation of the nucleoplasmic region of emerin before and after self-assembly, working on wild-type emerin as well as several mutants causing myopathies. I showed that the two fragments of emerin 1-187 and 67-221 were able to self-assemble, whereas their common region, 67-187, is always a monomer in our conditions (Samson et al., 2018). I also revealed that the LEM domain is at least partially unfolded during self-assembly of region 1-187, as a mutant with a destabilized LEM domain self-assembles faster and a mutant with a LEM domain locked in its folded conformation cannot self-assemble (Samson et al., 2017). I also assigned all the NMR signals of the unfolded region 67-170, in order to further study by NMR the impact of phosphorylation of this region on emerin structure and self-assembly properties (Samson et al., 2016). In the case of lamin A, I studied the C-terminal region of prelamin A that is predicted as unfolded and is heavily post-translationally modified. I assigned the NMR signals of this prelamin A peptide as well as its mutant peptide corresponding to the progerin sequence (Celli et al., 2018). I showed that both peptides are indeed unstructured and exhibit a partially populated  helix that has a highly conserved sequence. I propose that this helix is a binding site for a yet unidentified partner. I also revealed that the prelamin A peptide has a tendency to self-assemble. However, the monomeric prelamin A and progerin peptides, wild-type as well as farnesylated, do not interact with the immunoglobulin-like domain of lamin A/C and with BAF, two domains associated with progeria. Then, I investigated the interactions mediated by these peptides and two other important partners associated to progeria: the inner nuclear membrane SUN1 and the chromatin-associated protein RBBP4. However, SUN1 is also intrinsically disordered and poorly soluble in our conditions. First results showed that the prelamin peptide does not bind to RBBP4 but might need the remaining part of the lamin A tail for this interaction. However, RBBP4 directly binds to the lamin partner BAF. Based on my results, I propose a set of experiments to identify the molecular details of the interactions between the lamin A tail, BAF and RBBP4.

Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs), which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by γ-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of xeroderma pigmentosum group A (XPA), a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and coimmunoprecipitation. RNA interference (RNAi) knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.