Doctor of Philosophy / Graduate Biochemistry Group / Michal Zolkiewski / ClpB, a bacterial chaperone that belongs to the AAA+ protein family, cooperates with the Hsp70/40 system (DnaK, DnaJ and GrpE in E.coli) in the reactivation of aggregated substrates by translocating them through the central channel of its hexameric form. ClpB is essential for survival of bacteria under heat shock and plays an important role in the infectivity of pathogenic microorganisms. However the detailed mechanism of ClpB disaggregation activity is still not clear. ClpB is a multi-domain protein, which consists of two nucleotide binding domains (NBD1 and NBD2) connected by the middle domain (M domain), and the N-terminal domain connected to the rest of the protein by a flexible linker. In this work, mutations were introduced into the linker region to modify the mobility of the N-terminal domain. It was found that without altering the proper folding and oligomerization of ClpB, all the mutants had deficiencies in aggregate reactivation, possibly due to the weaker binding to aggregated substrates in the initial step of disaggregation. This led to the conclusion that the flexible attachment of the N-terminal domain supports substrate binding and controls the disaggregation by ClpB. Moreover, partial inhibition of the ClpB chaperone activity was observed for all the linker variants, suggesting that the linker sequence might have been optimized by selective pressure to maintain the optimal efficiency of aggregate reactivation. To study the substrate translocation of ClpB, a BAP (ClpB-ClpA P-loop) variant that binds to the protease ClpP was constructed. A FRET-based experiment was designed and the fluorescently-labeled ClpB substrates were produced. This work sets the stage for further studies on the mechanism of aggregate recognition by ClpB. ClpB also plays important roles in pathogenic bacteria invasion and virulence. Recombinant ClpB from Ehrlichia chaffeensis, a pathogenic bacterium that causes human monocytic ehrlichiosis, was purified to study its biochemical properties. Ehrlichia ClpB (Eh_B) and E.coli ClpB (Ec_B) sequences are highly conserved in the nucleotide binding region and poorly conserved in the N-terminal and M domain. The oligomerization, ATPase activity, chaperone activity and substrate binding of the recombinant Eh_B were tested. Recombinant Eh_B was able to reactivate aggregated proteins in the presence of HSP70 from E.coli with equal efficiency as Ec_B. However, the mechanism of Eh_B interactions with substrates and/or substrate specificity may be different from that of E. coli ClpB.

L’étude du microbiote intestinal et de ses produits de sécrétion est un domaine de recherche en expansion en raison des perspectives thérapeutiques que cela peut ouvrir pour les maladies nutritionnelles telles que l’obésité ou les TCA. La Caseinolytic peptidase B (ClpB) est une protéine bactérienne produite par les entérobactéries qui présente un mimétisme moléculaire avec l’α-MSH, un neuropeptide anorexigène signalant la satiété au niveau de l’hypothalamus. Des études récentes ont cherché à évaluer si la protéine ClpB pouvait induire des effets anorexigènes similaires à ceux de l’α-MSH, en situation physiopathologique et dans des modèles de troubles nutritionnels. Dans ce contexte, l’objectif de cette thèse a été d’étudier les potentiels effets pharmacologiques de la protéine ClpB, intacte ou fragmentée sur les différents mécanismes de régulation de la prise alimentaire impliquant l’axe microbiote-intestin-cerveau et l’influence des nutriments. La première étude a évalué in vitro l’impact de trois macronutriments sur la production et l’expression de la protéine ClpB par les bactéries E. coli : seul l’apport protéique augmentait significativement la production de ClpB. Nous avons montré que la ClpB augmentait la sécrétion de PYY par les cellules entéro-endocrines intestinales de rat en culture. La deuxième d’étude a été réalisée chez des souris dans un modèle d’anorexie (ABA). La restriction alimentaire, avec ou sans activité physique, augmentait la concentration plasmatique de ClpB, qui était associée à une augmentation de la proportion d’entérobactéries dans le microbiote, ce qui suggère son implication possible dans la physiopathologie de l’anorexie mentale. Enfin, la troisième étude a évalué in vivo chez le Rongeur, les effets pharmacologiques de la protéine ClpB sur la prise alimentaire. La prise alimentaire était réduite par l’injection de ClpB intacte ou fragmentée, mais pas par son fragment de 25 kDa. Ces résultats confortent le rôle de la protéine ClpB, produite par les entérobactéries, dans la régulation physiologique de la prise alimentaire et incitent à poursuivre l’étude de son implication dans les troubles anorexiques et son application thérapeutique dans les situations d’excès de poids. / The study of the gut microbiota and especially the effect of its secretory products is an expanding field of research in order to open therapeutic perspectives for nutritional diseases such as obesity or TCA. Among these molecules, the Caseinolytic peptidase B (ClpB) is a bacterial protein having a molecular mimicry in common with α-MSH, a neuropeptide whose anorectic actions are peripheral and central and possible via a microbiota-intestinal-brain communication. Current studies attempt to demonstrate whether this molecular mimicry can confer similar anorectic effects at the ClpB protein. The aim of this thesis was studied the potential pharmacological effects of ClpB protein the regulation of eating behavior. Given that the composition of the gut microbiota is dependent on the food present, the first study was evaluated in vitro the impact of three types of macronutrients on the production and expression of the ClpB protein by E. coli bacteria. Then, it was evaluated whether this protein could influence the secretion of satietogenic peptides like PYY by intestinal enteroendocrine cells using a primary culture of rat intestinal cells. Previous studies of U 1073 laboratory have shown that this protein has been found at the plasma level, the second study was performed in mice submitted to an anorexia model (ABA) to clarify the impact of dietary restriction on the ClpB protein, to better understand its possible involvement in the physiopathology of anorexia nervosa. Finally, the third study was evaluated the pharmacological effects of ClpB protein on food intake in vivo in rodents. The impact of the natural fragmentation of this protein and particularly of one of its fragments on food intake was also evaluated.

Master of Science / Department of Biochemistry and Molecular Biophysics / Michal Zolkiewski / ClpB is a molecular chaperone that is essential for infectivity and pathogen survival in a host. It belongs to the AAA+ protein family, which cooperates with the DnaK chaperone system to reactivate aggregated proteins. In this study, we purified and then studied the biochemical properties of the apicoplast targeted ClpB isoform from the malaria parasite Plasmodium falciparum: PfClpB1. Plasmodium falciparum is the parasite responsible for the most severe form of malaria. In contrast to the parasitophorous vacuole targeted PfClpB2 from Plasmodium falciparum which contains all characteristic AAA+ sequence motifs, PfClpB1 also includes a 52-residue long non-conserved insert in the middle domain. The ATPase activity study shows that PfClpB1 hydrolyzes ATP in presence of Poly-lysine and α-casein. Similar to most AAA+ ATPases, addition of ATP induces hexamer formation in PfClpB1. Lastly, PfClpB1 reactivates aggregated firefly luciferase. However, PfClpB1 is unable to efficiently reactivated luciferase in the presence of the E. coli DnaK chaperone system or human Hsp70 and Hsp40 (Hdj1). This can be explained by the extra middle domain sequence of PfClpB1. Our data may suggest that PfClpB1 activity is essential for Plasmodium falciparum survival by preserving the activity of apicoplast proteins.

The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants from heat stress. However, in contrast to the yeast ortholog, Hsp104, the C-terminal extension of HSP101 was found to be completely dispensable for thermotolerance of Arabidopsis. Additional transgenic plants expressing an HSP101-GFP were also characterized. Initial microscopic analysis confirms nuclear/cytoplasmic localization as has been reported previously for yeast Hsp104. However, the dynamics of subcellular redistribution upon heat stress need to be further investigated to fully understand the potential significance of the observed localization.

No description available.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-25T12:11:00Z No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-25T12:11:00Z (GMT). No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-08 / Acinetobacter baumannii (A. baumannii) is an important opportunistic, Gram-negative pathogen responsible for severe nosocomial infections such as pneumonia, septicemia, urinary tract infections and meningitis. Strains of A. baumannii have been identified in an endemic and epidemic manner in hospitals, being verified the occurrence of multiresistant strains in these environments, with important ability to adapt to selective changes and environmental pressures. Furthermore, multidrug resistance to antibiotics has been continuously studied because it is a global public health problem, resulting in failure of therapy, prolongation of hospitalization, increase of mortality and morbidity rates, and increase in the financial costs of treatment. This pathogen has varied strategies involved with antimicrobial resistance, but it is known that the bacteria are able to respond to unfavorable conditions in the medium through the rapid expression of heat shock proteins (HSP) and also appear to be involved with the stress response caused by the presence of antibiotics. Among the HSPs is ClpB, an ATP-dependent molecular chaperone belonging to the HSP100 family that is associated with several cellular activities, with the remarkable ability to rescue proteins damaged by stress. The objective of this work was to investigate the role of the clpB gene responsible for the coding of a heat shock protein through qPCR in response to stress generated by thermal shock and antibiotics in cells of a multidrug resistant strain of A. baumannii (RS4). Tests performed included analysis of the structure of the clpB gene with bioinformatics tools and analysis of the expression of the same gene by qRT-PCR in response to exposure to heat shock and subinhibitory concentrations of the following antibiotics: ampicillin (30 g mL-1 ), amoxicillin + sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfamethoxazole + trimethoprim (120/8 g mL-1) and meropenem (18 g mL-1).The analysis of the qPCR results showed a transient increase in the induction of the clpB gene in the different treatments used in this study and repression of mRNA-clpB in the presence of cefepime. In addition, in the presence of ampicillin and amoxicillin associated with sulbactam the increase in mRNA-clpB synthesis was around 1.4 times higher after 20 min of incubation with the antibiotics than in the complete absence of the antibiotics. Surprisingly, in the presence of meropenen the induction of mRNA-clpB expression was more than 30-fold higher after 10 minutes of incubation with the antibiotic and more than 8-fold higher in the presence of sulfamethoxazole associated with trimetropin. These data suggest that A. baumannii through thermal stress and antibiotic exposure, adjusts transcription levels of gene clpB allowing the bacterium to survive unfavorable conditions of the medium. Consequently, it can be stated that the protein encoded by the clpB gene is an important virulence factor in response to antibiotics in this pathogen. / Acinetobacter baumannii (A. baumannii) é um importante patógeno oportunista, Gram-negativo e responsável por infecções nosocomiais severas como pneumonias, septicemias, infecções urinárias e meningites. Cepas de A. baumannii têm sido identificadas de maneira endêmica e epidêmica nos hospitais, sendo verificada a ocorrência de cepas multirresistentes nesses ambientes, com importante habilidade de adaptação a mudanças seletivas e pressões ambientais. Ainda, a multirresistência a antibióticos vem sendo estudada continuamente, por se tratar de um problema de saúde pública global, resultando em falha na terapia, no prolongamento da internação hospitalar, no aumento das taxas de mortalidade e morbidade e na elevação dos custos financeiros do tratamento. Esse patógeno possui estratégias variadas envolvidas com a resistência aos antimicrobianos, porém, sabe-se que as bactérias apresentam habilidade de responder a condições desfavoráveis do meio em que se encontram por meio da rápida expressão de proteínas de choque térmico (HSP) e parecem também estar envolvidas com a resposta a estresse causado pela presença de antibióticos. Dentre as HSPs, está a ClpB, chaperone molecular dependente de ATP, pertencente à família HSP100 que está associada a diversas atividades celulares, com a capacidade notável para resgatar proteínas danificadas pelo estresse. O objetivo deste trabalho foi investigar o papel do gene clpB em resposta a estresse gerado por choque térmico e antibióticos em células de uma cepa multirresistente de A. baumannii (RS4). Os testes realizados englobaram análise da estrutura do gene clpB com ferramentas de bioinformática e análise da expressão do mesmo gene por qRT-PCR em resposta à exposição a choque térmico e a concentrações subinibitórias dos seguintes antibióticos: ampicilina (30 g mL-1), amoxacilina+ sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfametoxazol + trimetoprima (120/8 g/mL-1) e meropenem (18 g mL-1). Os resultados apontados por análise de bioinformática sugerem uma conservação da estrutura global de ClpB dentro do gênero Acinetobacter sp. A análise dos resultados de qPCR evidenciou aumento transitório na indução do gene clpB nos diferentes tratamentos utilizados neste estudo e repressão do mRNA-clpB na presença de cefepime. Em adição, tanto na presença de ampicilina como de amoxicilina associada à sulbactam o aumento na síntese de mRNA-clpB foi em torno de 1,4 vezes superior após 20 min de incubação com os antibióticos do que na completa ausência dos antibióticos. Surpreendentemente, na presença de meropenen, a indução da expressão do mRNA-clpB foi mais que 30 vezes superior após 10 minutos de incubação com o antibiótico e mais que 8 vezes superior na presença de sulfametoxaxol associado à trimetropina. Esses dados sugerem que A. baumannii, mediante estresse térmico e exposição a antibióticos, ajusta os níveis de transcrição do gene clpB, permitindo que a bactéria sobreviva a condições desfavoráveis do meio. Consequentemente, pode-se afirmar que a proteína codificada pelo gene clpB figura como importante fator de virulência em resposta a antibióticos neste patógeno.

La déficience intellectuelle (DI) définit un groupe de conditions génétiquement hétérogènes caractérisées par l’apparition de troubles cognitifs précoces chez l’enfant. Elle affecte 1-3% de la population dans les pays industrialisés. La prévalence de la DI est beaucoup plus élevée ailleurs dans le monde, en raison de facteurs sociodémographiques comme le manque de ressources dans le système de santé, la pauvreté et la consanguinité. Des facteurs non-génétiques sont mis en cause dans l’étiologie de la DI ; on estime qu’environ 25% des cas de DI sont d’origine génétique. Traditionnellement, les bases moléculaires de la DI ont été investiguées par des analyses cytogénétiques, les approches de cartographie génétique et le séquençage de gènes candidats ; ces techniques de génétiques classiques sont encore mises à rude épreuve dans l’analyse de maladies complexes comme la DI. La DI liée à l’X a été particulièrement étudiée, avec plus d’une centaine de gènes identifiés uniquement sur le chromosome X. Des mutations hétérozygotes composites sont mises en évidence dans la DI autosomique, dans le contexte d’unions non-consanguines. L’occurrence de ce type de mutations est rare, chez des individus non-apparentés, de sorte que les mutations dominantes de novo sont plus courantes. Des mutations homozygotes sont attendues dans les populations consanguines ou marquées par un effet fondateur. En fait, les bases moléculaires de la DI autosomique ont été presqu’exclusivement étudiées dans le contexte de populations avec des forts taux de consanguinité. L’origine de la DI demeure encore inconnue dans environ 60 % des cas diagnostiqués. En l’absence de facteurs environnementaux associés à la DI chez ces individus, il est possible d’envisager que des facteurs génétiques non identifiés entrent en jeu dans ces cas de DI inexpliqués. Dans ce projet de recherche, nous voulions explorer l’origine génétique de la DI, dans vingt familles, où une transmission de la maladie selon un mode autosomique récessif est suspectée. Nous avons mis de l’avant les techniques de séquençage de nouvelle génération, afin de mettre en évidence les déterminants génétiques de la DI, à l’échelle du génome humain. En fait, nous avons priorisé la capture et le séquençage de l’exome; soient la totalité des régions codantes du génome humain et leurs sites d’épissage flanquants. Dans nos analyses, nous avons ciblé les variants qui ne sont pas rapportés trop fréquemment dans différentes bases de données d’individus contrôles, ces mutations rares cadrent mieux avec une condition comme la DI. Nous avons porté une attention particulière aux mutations autosomiques récessives (homozygotes et hétérozygotes composites) ; nous avons confirmé que ces mutations ségréguent avec une transmission récessive dans la famille à l’étude. Nous avons identifié des mutations dans des gènes pouvant être à l’origine de la DI, dans certaines des familles analysées ; nous avons validé biologiquement l'impact fonctionnel des mutations dans ces gènes candidats, afin de confirmer leur implication dans la pathophysiologie de la DI. Nous avons élucidé les bases moléculaires de la DI dans huit des familles analysées. Nous avons identifié le second cas de patients avec syndrome de cassure chromosomique de Varsovie, caractérisé par des dysfonctions de l’ARN hélicase DDX11. Nous avons montré qu’une perte de l’activité de TBC1D7, une des sous-unités régulatrice du complexe TSC1-TSC2, est à l’origine de la pathologie dans une famille avec DI et mégalencéphalie. Nous avons mis en évidence des mutations pathogéniques dans le gène ASNS, codant pour l’Asparagine synthétase, chez des patients présentant une microcéphalie congénitale et une forme progressive d’encéphalopathie. Nous avons montré que des dysfonctions dans la protéine mitochondriale MAGMAS sont mises en cause dans une condition caractérisée par un retard prononcé dans le développement associé à une forme sévère de dysplasie squelettique. Nous avons identifié une mutation tronquant dans SPTBN2, codant pour la protéine spinocerebellar ataxia 5, dans une famille avec DI et ataxie cérébelleuse. Nous avons également mis en évidence une mutation dans PIGN, un gène impliqué dans la voie de biosynthèse des ancres de glycosylphosphatidylinositol , pouvant être à l’origine de la maladie chez des individus avec épilepsie et hypotonie. Par ailleurs, nous avons identifié une mutation - perte de fonction dans CLPB, codant pour une protéine chaperonne mitochondriale, dans une famille avec encéphalopathie néonatale, hyperekplexie et acidurie 3-méthylglutaconique. Le potentiel diagnostic des techniques de séquençage de nouvelle génération est indéniable ; ces technologies vont révolutionner l’univers de la génétique moléculaire, en permettant d’explorer les bases génétiques des maladies complexes comme la DI. / Intellectual disability (ID) regroups greatly heterogeneous conditions that are characterized by early-onset cognitive impairment. ID affects about 1-3% of Western populations; but its prevalence is much higher in deprived regions of the world where socio-demographic factors like poor healthcare, lack of resources and parental consanguinity prevail. Non-genetic factors are involved in the etiology of ID; approximately 25% of ID cases are of genetic origin. Traditionally, the molecular basis of ID have been assessed through cytogenetic analyses, genetic mapping and candidate gene approaches. These classical genetic tools are still put to the test in the study of complex diseases like ID. Until recently, X-linked ID cases were the main focus of studies on ID with more than hundred ID genes identified only on the X chromosome. Compound heterozygous mutations are identified in autosomal forms of ID, in the context of non-consanguineous unions. However, the occurrence of such mutations is rare in outbred populations, so that dominant de novo mutations are most common in unrelated individuals. Homozygous mutations are expected in consanguineous unions or in populations marked by a founder effect. In fact, the molecular bases of autosomic recessive ID have been almost exclusively studied in populations with high consanguinity rates. ID remains unsolved in more than 60% of patients. In the absence of environmental factors associated with ID in these individuals, it is possible to consider that unidentified genetic factors are involved in these unexplained ID cases. In this research project, we used next generation sequencing technologies to highlight the genetic causes of ID in twenty families were an autosomal recessive mode of inheritance is expected. We prioritized the use of whole-exome sequencing, namely all coding exons in the genome of this individual. In our analyses, we filtered out variants that were too common in control individuals to describe a rare condition like ID. We focussed our attention on rare autosomic recessive varaiants (homozygous and compound heterozygous), these mutations were confirmed by Sanger re-sequencing to segregate with an autosomal recessive mode of inheritance in the family. We identified mutations in candidate genes for ID in some of the family analysed, we validated the functional impact of the mutations in these genes to confirm their involvement in the pathophysiology of ID in the family studied. We explained the molecular basis of ID in eight of the families studied. We identified the second case of Warsaw-Breakage-Syndrome, a rare genetic disorder characterised by dysfunction of the RNA helicase DDX11. We showed that disruption in TBC1D7, a functional subunit of the TSC1-TSC2 protein complex, cause ID and megalencephaly. We demonstrated that ASNS, the Asparagine Synthetase gene, is defective in patients with congenital microcephaly and progressive encephalopathy. We showed that the gene coding for the mitochondrial protein MAGMAS is involved in the pathophysiology of a condition characterised by developmental delay and severe skeletal dysplasia. We identified a truncating mutation in SPTBN2, encoding for the spinocerebellar ataxia 5 proteins, in a family with ID and spinocerellar ataxia. We also identified a mutation in a gene involved in the biosynthetic pathway of glycosylphosphatidylinositol anchors; the mutation in PIGN may cause the epilepsy and hypotonia features observed in the affected individuals of that family. Finally, we identified a loss of function mutation in CLPB, coding for a mitochondrial chaperone, in individuals with severe encephalopathy, hypereklexia and 3-methylglutaconic aciduria. The diagnostic potential of next generation sequencing technologies is undeniable. These technologies will revolutionize the world of molecular genetics; they will help deciphering the molecular basis of complex diseases like ID.