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A key prerequisite for cellular and organismal health is a functional proteome. A variety of human protein misfolding diseases are associated with the occurrence of amyloid protein aggregates, such as amyotrophic lateral sclerosis (ALS) or Huntington’s disease. The proteins involved in disease manifestation all contain aggregation-prone sequences of low compositional complexity. Such sequences are also known as prion-like, because of their sequence similarity to yeast prions. Yeast prion proteins are a specific subset of amyloid forming proteins with distinct physio-chemical and functional features, which give them transmissible properties. The aggregation properties of yeast prions and disease-related prion-like proteins reside in structurally independent, prion-forming domains (PrDs). These domains are highly enriched for uncharged polar amino acids, such as glutamine (Q) and asparagine (N). These compositional features can be used to predict prion-like proteins bioinformatically. To investigate the prevalence of prion-like proteins across different organisms, we analyzed a range of eukaryotic proteomes. Our analysis revealed that the slime mold D. discoideum contains the highest number of prion-like N/Q-rich proteins of all organisms. Based on this finding, we hypothesized that D. discoideum could be a valuable model system to study protein homeostasis (proteostasis) and the molecular basis of protein misfolding diseases. To explore how D. discoideum manages its highly aggregation-prone proteome, we analyzed the behavior of several well-characterized misfolding-prone marker proteins (variants of the disease-causing exon 1 of the huntingtin protein as well as wildtype and variant versions of the Q/N-rich yeast prion Sup35NM). Intriguingly, these proteins did not form cytosolic aggregates in D. discoideum, as they do in other organisms. Aggregates, however, formed as a result of heat stress, which indicates that the tested proteins have the capacity to aggregate, but are kept under tight control under normal conditions. Furthermore, when the stress level was reduced, the stress-induced aggregates dissolved, suggesting that D. discoideum has evolved mechanisms to reverse aggregation after a period of acute stress. Together, these findings reveal an unusual resilience of D. discoideum to aggregation-prone proteins, which very likely results from specific adaptations in its proteostasis network. By studying these specific adaptations, we could get important insight into the strategies that nature employs to control and maintain a highly aggregation-prone proteome. So far, our experimental investigations have revealed evidence for three specific adaptations. First, we identified the disaggregase Hsp101 as a key player in the acute stress response of D. discoideum. A functional analysis of Hsp101 in yeast and D. discoideum revealed that it supports thermotolerance. Second, we found evidence for an important role of the nucleus and nucleolus in proteostasis. We discovered that a small fraction of highly aggregation-prone proteins accumulated in the nucleus or nucleolus of D. discoideum cells. The magnitude of this nuclear accumulation could be increased by proteasome impairment, which suggests that the ubiquitin-proteasome system (UPS) is involved. This finding is consistent with previous studies in other organisms and hints at the possibility that D. discoideum disposes of aggregation-prone proteins by degrading them in the nucleus/nucleolus. Third and finally, we found that cells containing nuclear accumulations are asymmetrically distributed in the multicellular developmental stage (slug), suggesting that D. discoideum employs cell-sorting mechanisms to dispose of cells with accumulated protein damage. Although our current understanding of proteostasis in D. discoideum is preliminary, we have gained important insight into the molecular mechanisms and cellular pathways that D. discoideum uses to counteract protein aggregation. Findings from this work will inform similar comparative studies in other organisms and will impact our molecular understanding of protein misfolding diseases and aging. / Eine wesentliche Voraussetzung für die Gesundheit von Zellen und Organismen ist ein funktionales Proteom. Eine Reihe von humanen Protein- Missfaltungs-Erkrankungen, wie Chorea Huntington und Amyotrophe Lateralsklerose (ALS) werden mit dem Auftreten von amyloiden Protein- Aggregaten in Verbindung gebracht. Sämtliche Proteine, die in der Pathogenese dieser Krankheiten eine Rolle spielen, enthalten aggregations-anfällige Sequenzen mit geringer Sequenzkomplexität. Solche Sequenzen werden als Prion-ähnlich bezeichnet, da sie in ihrer Zusammensetzung den Prionen aus der Hefe S. cerevisiae gleichen. Die Prion-Proteine der Hefe gehören zu einer Unterart von amyloid-aggregierenden Proteinen, die durch bestimmte physikochemische und funktionelle Eigenschaften einen infektiösen Charakter erhalten. Die Aggregations-Eigenschaften von Hefeprionen und aggregationsanfällige Proteinen, die mit Erkrankungen in Verbindung gebracht werden, basieren auf strukturell unabhängigen, Prion-bildenden Domänen (prion domain, PrD). Diese Domänen sind angereichert mit polaren Aminosäuren wie Glutamin und Asparagin. Diese Zusammensetzung kann dazu verwendet werden prion-ähnliche Proteine bioinformatisch vorherzusagen. Um die Verbreitung von Prion-ähnlichen Proteinen in verschiedenen Organismen zu untersuchen, analysierten wir eine Reihe von eukaryotischen Proteomen. Unsere Analyse zeigte, dass der Schleimpilz D. discoideum die höchste Anzahl von Prion-ähnlichen N/Q-reichen Proteinen aufzeigt. Aufgrund dieser Erkenntnisse erstellten wir die Hypothese, dass D. discoideum ein nützlicher Modellorganismus sein könnte, um Protein Homöostase (Proteostase) sowie die molekulare Basis von Proteins-Missfaltungs-Erkrankungen zu ergründen. Um zu analysieren, wie D. discoideum mit seinem höchst aggregations-anfälligen Proteom umgehen kann, untersuchten wir das Verhalten mehrerer bereits charakterisierter aggregations-anfälliger Marker-Proteine in D. discoideum. Hierbei verwendeten wir Varianten des krankheits-erzeugenden Exon 1 des humanen Huntingtin Protein sowie den wild-typ und Varianten des N/Q-reichen Hefe Prions Sup35. Interessanterweise bildeten diese Proteine, anders als in anderen Organismen, keine zytosolischen Aggregate in D. discoideum aus. Aggregate wurden jedoch unter Hitzestress-Bedingungen gebildet. Dies deutet darauf hin, dass die getesteten Proteine durchaus das Vermögen zu aggregieren besitzen, jedoch unter normalen Wachstumsbedingungen streng kontrolliert werden. Wenn, darüberhinaus das Stress- Level gesenkt wurde, kam es zur Auflösung der stress-induzierten Aggregate. Dies deutet darauf hin, dass D. discoideum Mechanismen entwickelt hat, um Aggregate nach Perioden von akutem Stress wieder aufzulösen. Zusammengenommen enthüllen diese Erkenntnisse eine ungewöhnliche Widerstandsfähigkeit gegenüber aggregations-anfälligen Proteinen. Diese beruht höchstwahrscheinlich auf spezifischen Modifikationen im Proteostase Netzwerk. Durch die Analyse dieser spezifischen Anpassungen könnten wichtige Einblicke in die Strategien gewährt werden, welche die Natur benutzt, um ein höchst aggregations-anfälliges Proteom zu erhalten und zu kontrollieren. Bisher erbrachten unsere Experimente Anhaltspunkte für drei spezifische Anpassungen. Erstens zeigten wir, dass die Disaggregase Hsp101 eine Schlüsselrolle in der akuten Stressantwort in D. discoideum einnimmt. Eine funktionale Analyse von Hsp101 in D. discoideum und Hefe zeigte, dass die Disaggregase Thermotoleranz fördert. Zweitens haben wir Anhaltspunkte, dass der Nukleus und der Nukleolus eine wichtige Rolle in der Proteostase einnehmen. Eine geringe Fraktion der überaus aggregations-anfälligen Proteine akkumuliert im Nukleus oder Nukleolus von D. discoideum. Das Ausmaß der nuklearen Akkumulation konnte erhöht werden, wenn das Proteasom beeinträchtigt wird. Dies deutet darauf hin, dass das Ubiquitin-Proteasom-System involviert sein könnte. Diese Beobachtung ist im Einklang mit jüngsten Berichten aus anderen Organismen und daraus folgt, dass D. discoideum möglicherweise aggregations-anfällige Proteine durch Abbau im Nukleus entsorgt. Drittens konnten wir feststellen, dass Zellen, die nukleare Akkumulationen enthalten, asymmetrisch in der multizellulären Entwicklungs-Struktur des Pseudoplasmodiums verteilt sind. Dies deutet darauf hin, dass D. discoideum möglicherweise den Zellsortierungsmechanismus während der Entwicklung nutzen kann, um Zellen mit angereicherten Protein-Schäden zu beseitigen. Auch wenn das gegenwärtige Verständnis der Proteostase in D. discoideum nur vorläufig ist, haben wir wichtige Einblicke in die molekularen Mechanismen und zellulären Prozesse erhalten, die D. discoideum verwendet, um Protein-Aggregation zu verhindern. Die Ergebnisse dieser Arbeit werden ähnliche vergleichende Studien in anderen Organismen beeinflussen und Auswirkungen auf unser molekulares Verständnis über Protein-Missfaltungs-Erkrankungen und das Altern haben.

A key prerequisite for cellular and organismal health is a functional proteome. A variety of human protein misfolding diseases are associated with the occurrence of amyloid protein aggregates, such as amyotrophic lateral sclerosis (ALS) or Huntington’s disease. The proteins involved in disease manifestation all contain aggregation-prone sequences of low compositional complexity. Such sequences are also known as prion-like, because of their sequence similarity to yeast prions. Yeast prion proteins are a specific subset of amyloid forming proteins with distinct physio-chemical and functional features, which give them transmissible properties. The aggregation properties of yeast prions and disease-related prion-like proteins reside in structurally independent, prion-forming domains (PrDs). These domains are highly enriched for uncharged polar amino acids, such as glutamine (Q) and asparagine (N). These compositional features can be used to predict prion-like proteins bioinformatically. To investigate the prevalence of prion-like proteins across different organisms, we analyzed a range of eukaryotic proteomes. Our analysis revealed that the slime mold D. discoideum contains the highest number of prion-like N/Q-rich proteins of all organisms. Based on this finding, we hypothesized that D. discoideum could be a valuable model system to study protein homeostasis (proteostasis) and the molecular basis of protein misfolding diseases. To explore how D. discoideum manages its highly aggregation-prone proteome, we analyzed the behavior of several well-characterized misfolding-prone marker proteins (variants of the disease-causing exon 1 of the huntingtin protein as well as wildtype and variant versions of the Q/N-rich yeast prion Sup35NM). Intriguingly, these proteins did not form cytosolic aggregates in D. discoideum, as they do in other organisms. Aggregates, however, formed as a result of heat stress, which indicates that the tested proteins have the capacity to aggregate, but are kept under tight control under normal conditions. Furthermore, when the stress level was reduced, the stress-induced aggregates dissolved, suggesting that D. discoideum has evolved mechanisms to reverse aggregation after a period of acute stress. Together, these findings reveal an unusual resilience of D. discoideum to aggregation-prone proteins, which very likely results from specific adaptations in its proteostasis network. By studying these specific adaptations, we could get important insight into the strategies that nature employs to control and maintain a highly aggregation-prone proteome. So far, our experimental investigations have revealed evidence for three specific adaptations. First, we identified the disaggregase Hsp101 as a key player in the acute stress response of D. discoideum. A functional analysis of Hsp101 in yeast and D. discoideum revealed that it supports thermotolerance. Second, we found evidence for an important role of the nucleus and nucleolus in proteostasis. We discovered that a small fraction of highly aggregation-prone proteins accumulated in the nucleus or nucleolus of D. discoideum cells. The magnitude of this nuclear accumulation could be increased by proteasome impairment, which suggests that the ubiquitin-proteasome system (UPS) is involved. This finding is consistent with previous studies in other organisms and hints at the possibility that D. discoideum disposes of aggregation-prone proteins by degrading them in the nucleus/nucleolus. Third and finally, we found that cells containing nuclear accumulations are asymmetrically distributed in the multicellular developmental stage (slug), suggesting that D. discoideum employs cell-sorting mechanisms to dispose of cells with accumulated protein damage. Although our current understanding of proteostasis in D. discoideum is preliminary, we have gained important insight into the molecular mechanisms and cellular pathways that D. discoideum uses to counteract protein aggregation. Findings from this work will inform similar comparative studies in other organisms and will impact our molecular understanding of protein misfolding diseases and aging. / Eine wesentliche Voraussetzung für die Gesundheit von Zellen und Organismen ist ein funktionales Proteom. Eine Reihe von humanen Protein- Missfaltungs-Erkrankungen, wie Chorea Huntington und Amyotrophe Lateralsklerose (ALS) werden mit dem Auftreten von amyloiden Protein- Aggregaten in Verbindung gebracht. Sämtliche Proteine, die in der Pathogenese dieser Krankheiten eine Rolle spielen, enthalten aggregations-anfällige Sequenzen mit geringer Sequenzkomplexität. Solche Sequenzen werden als Prion-ähnlich bezeichnet, da sie in ihrer Zusammensetzung den Prionen aus der Hefe S. cerevisiae gleichen. Die Prion-Proteine der Hefe gehören zu einer Unterart von amyloid-aggregierenden Proteinen, die durch bestimmte physikochemische und funktionelle Eigenschaften einen infektiösen Charakter erhalten. Die Aggregations-Eigenschaften von Hefeprionen und aggregationsanfällige Proteinen, die mit Erkrankungen in Verbindung gebracht werden, basieren auf strukturell unabhängigen, Prion-bildenden Domänen (prion domain, PrD). Diese Domänen sind angereichert mit polaren Aminosäuren wie Glutamin und Asparagin. Diese Zusammensetzung kann dazu verwendet werden prion-ähnliche Proteine bioinformatisch vorherzusagen. Um die Verbreitung von Prion-ähnlichen Proteinen in verschiedenen Organismen zu untersuchen, analysierten wir eine Reihe von eukaryotischen Proteomen. Unsere Analyse zeigte, dass der Schleimpilz D. discoideum die höchste Anzahl von Prion-ähnlichen N/Q-reichen Proteinen aufzeigt. Aufgrund dieser Erkenntnisse erstellten wir die Hypothese, dass D. discoideum ein nützlicher Modellorganismus sein könnte, um Protein Homöostase (Proteostase) sowie die molekulare Basis von Proteins-Missfaltungs-Erkrankungen zu ergründen. Um zu analysieren, wie D. discoideum mit seinem höchst aggregations-anfälligen Proteom umgehen kann, untersuchten wir das Verhalten mehrerer bereits charakterisierter aggregations-anfälliger Marker-Proteine in D. discoideum. Hierbei verwendeten wir Varianten des krankheits-erzeugenden Exon 1 des humanen Huntingtin Protein sowie den wild-typ und Varianten des N/Q-reichen Hefe Prions Sup35. Interessanterweise bildeten diese Proteine, anders als in anderen Organismen, keine zytosolischen Aggregate in D. discoideum aus. Aggregate wurden jedoch unter Hitzestress-Bedingungen gebildet. Dies deutet darauf hin, dass die getesteten Proteine durchaus das Vermögen zu aggregieren besitzen, jedoch unter normalen Wachstumsbedingungen streng kontrolliert werden. Wenn, darüberhinaus das Stress- Level gesenkt wurde, kam es zur Auflösung der stress-induzierten Aggregate. Dies deutet darauf hin, dass D. discoideum Mechanismen entwickelt hat, um Aggregate nach Perioden von akutem Stress wieder aufzulösen. Zusammengenommen enthüllen diese Erkenntnisse eine ungewöhnliche Widerstandsfähigkeit gegenüber aggregations-anfälligen Proteinen. Diese beruht höchstwahrscheinlich auf spezifischen Modifikationen im Proteostase Netzwerk. Durch die Analyse dieser spezifischen Anpassungen könnten wichtige Einblicke in die Strategien gewährt werden, welche die Natur benutzt, um ein höchst aggregations-anfälliges Proteom zu erhalten und zu kontrollieren. Bisher erbrachten unsere Experimente Anhaltspunkte für drei spezifische Anpassungen. Erstens zeigten wir, dass die Disaggregase Hsp101 eine Schlüsselrolle in der akuten Stressantwort in D. discoideum einnimmt. Eine funktionale Analyse von Hsp101 in D. discoideum und Hefe zeigte, dass die Disaggregase Thermotoleranz fördert. Zweitens haben wir Anhaltspunkte, dass der Nukleus und der Nukleolus eine wichtige Rolle in der Proteostase einnehmen. Eine geringe Fraktion der überaus aggregations-anfälligen Proteine akkumuliert im Nukleus oder Nukleolus von D. discoideum. Das Ausmaß der nuklearen Akkumulation konnte erhöht werden, wenn das Proteasom beeinträchtigt wird. Dies deutet darauf hin, dass das Ubiquitin-Proteasom-System involviert sein könnte. Diese Beobachtung ist im Einklang mit jüngsten Berichten aus anderen Organismen und daraus folgt, dass D. discoideum möglicherweise aggregations-anfällige Proteine durch Abbau im Nukleus entsorgt. Drittens konnten wir feststellen, dass Zellen, die nukleare Akkumulationen enthalten, asymmetrisch in der multizellulären Entwicklungs-Struktur des Pseudoplasmodiums verteilt sind. Dies deutet darauf hin, dass D. discoideum möglicherweise den Zellsortierungsmechanismus während der Entwicklung nutzen kann, um Zellen mit angereicherten Protein-Schäden zu beseitigen. Auch wenn das gegenwärtige Verständnis der Proteostase in D. discoideum nur vorläufig ist, haben wir wichtige Einblicke in die molekularen Mechanismen und zellulären Prozesse erhalten, die D. discoideum verwendet, um Protein-Aggregation zu verhindern. Die Ergebnisse dieser Arbeit werden ähnliche vergleichende Studien in anderen Organismen beeinflussen und Auswirkungen auf unser molekulares Verständnis über Protein-Missfaltungs-Erkrankungen und das Altern haben.

Doctor of Philosophy / Department of Biochemistry / Michal Zolkiewski / ClpB is a bacterial heat-shock protein that disaggregates and reactivates strongly aggregated proteins in cooperation with the DnaK chaperone system. ClpB contains two ATP-binding AAA+ modules, a linker coiled-coil domain, and a highly mobile N-terminal domain. It forms ring-shaped hexamers in a nucleotide-dependent manner. The unique aggregation reversing chaperone activity of ClpB involves ATP-dependent translocation of substrates through the central channel in the ClpB ring. The initial events of aggregate recognition and the events preceding the translocation step are poorly understood. In addition to the full-length ClpB95, a truncated isoform ClpB80, that is missing the whole N-terminal domain, is also produced in vivo. Various aspects of the structure and function of ClpB were addressed in this work. The thermodynamic stability of ClpB in its monomeric and oligomeric forms, as well as the nucleotide-induced conformational changes in ClpB were investigated by fluorescence spectroscopy. Equilibrium urea-induced unfolding showed that two structural domains-the small domain of the C-terminal AAA+ module and the coiled-coil domain-were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation or interactions during formation of the ClpB rings. Several locations of Trp-fluorescence probes were also found to respond to nucleotide binding. The biological role of the two naturally-occurring ClpB isoforms was also investigated. We discovered that ClpB achieves optimum chaperone activity by synergistic cooperation of the two isoforms that form hetero-oligomers. We found that ClpB95/ClpB80 hetero-oligomers form preferentially at low protein concentration with higher affinity than homo-oligomers of ClpB95. Moreover, hetero-oligomers bind to aggregated substrates with a similar efficiency as homo-oligomers of ClpB95, do not show enhanced ATPase activity over that of the homo-oligomers, but display a strongly stimulated chaperone activity during the reactivation of aggregated proteins. We propose that extraction of single polypeptides from aggregates and their delivery to the ClpB channel for translocation is the rate-limiting step in aggregate reactivation and that step is supported by the mobility of the N-terminal domain of ClpB. We conclude that the enhancement of the chaperone activity of the hetero-oligomers is linked to an enhancement of mobility of the N-terminal domains.

Protein solubility plays an important role whether for biophysical and structural studies, or for production and delivery of therapeutic proteins. Poor solubility could lead to protein aggregation, which is an undesired physicochemical mechanism at any stage of recombinant proteins production. To date, more than half of all recombinant therapeutic proteins are produced in mammalian cells, mainly due to the high similarity of the final product to human protein structures. However, poor secretion can occur, due to misfolded proteins or aggregates leading to cellular stress and proteolysis. Another widely-used expression system is E. coli, which can offer a cost-efficient alternative. This system has an important limitation, since proteins tends to form insoluble protein aggregates in the cytoplasm upon heterologous overexpression. Several strategies are being implemented to improved soluble expression, ranging from culture conditions to solubility enhancing tags. However, there is no universal approach or technology that solves protein aggregation. In this thesis two recently published hypotheses from our group have been applied. One stated that soluble expression of proteins was inversely correlated with the size of the largest positively-charged patch on the protein surface. The second hypothesis (of protein solubility), arose from the finding that the relative content of lysine and arginine residues separated E. coli proteins by solubility. Both hypotheses arose from a study of an extensive dataset of experimental solubilities determined for cell-free expression of E. coli proteins. In combination with other widely used strategies, such as lowering expression temperature and inducer concentration, decreasing non-charged (hydrophobic) patches and addition of helical capping for increasing stability, a rational understanding for directed alteration of solubility in a variety of recombinant proteins has been explored. This includes three protein models to test: (i) recombinant human erythropoietin (rHuEPO) (one of the top selling therapeutics) (ii) recombinant 6-Phosphofructo-2-Kinase/fructose-2,6-bisphosphatase (rPFKFB3) (a product for which over-expression has been sought for characterisation and insight into possible cancer therapy) and (iii) a set of three selected E. coli proteins containing high ratios of lysines to arginines: thioredoxin-1 (TRX), cold shock-like protein cspB (cspB), and the histidine-containing phosphocarrier protein (HPr). It was found that single or multiple point mutations (changing amino acids from positive to negative charge or vice versa; or lysines to arginines) verified the predicted effect on rHuEPO, rPFKFB3, TRX, cspB, and HPr solubility (experimentally defined as the distribution between soluble and total fractions) for expression in E. coli. In addition, the redesigned set of rHuEPO transiently expressed in HEK 293-EBNA cells, suggesting that positively-charged patch size may also influence protein secretion. Further application of these computational and experimental approaches could provide a valuable tool in the design and engineering of proteins, with enhanced solubility, stability and secretion.

La réponse au choc thermique est un mécanisme de défense largement décrit au cours duquel l’expression préférentielle des protéines de choc thermique Hsp aide la cellule à récupérer des dommages causés par l’hyperthermie, comme la dénaturation/agrégation des protéines. Une des conséquences du choc thermique mise en évidence au laboratoire, est l’activation du facteur de transcription NFκB. Cette activation a lieu pendant la période de récupération suivant ce stress. Par comparaison de la réponse au choc thermique de cellules témoins ou déficientes en NFκB, nous avons cherché à étudier les conséquences de l’activation de NFκB par le choc thermique. Nous avons montré que NFκB active un mécanisme augmentant la survie des cellules soumises à une hyperthermie : l’autophagie. L’absence d’induction de ce mécanisme conduit à la mort par nécrose des cellules déplétées en NFκB. Dans ces cellules, l’induction artificielle de l’autophagie restaure une survie normale au stress thermique. Nous avons montré que les principaux régulateurs de l’autophagie (complexes mTOR et PI3Kinase de Classe III) ne sont pas des cibles modulées par NFκB, en réponse à une hyperthermie. En revanche, l’accumulation de protéines dénaturées voire agrégées est un élément primordial pour l’activation de l’autophagie-dépendante de NFκB. En effet dans les cellules déficientes pour NFκB, contrairement aux cellules témoins, l’accumulation de protéines agrégées induite par le traitement hyperthermique, mais aussi par l’expression de formes mutées d’HspB5, n’est pas résorbée ; ceci indique que le contrôle qualité des protéines est altéré dans ces cellules. Cette altération pourrait provenir d’un défaut de formation du complexe BAG3-HspB8 en absence de NFκB. En effet, nous avons montré que la forte expression des gènes bag3 et hspb8, induite suite au stress thermique, est dépendante de NFκB et que l’accumulation du complexe BAG3-HspB8, observé dans les cellules témoins soumises au choc thermique, est inhibée dans les cellules déficientes pour NFκB. Nos résultats démontrent que NFκB induit un processus autophagique en réponse à l’agrégation protéique induite par l’hyperthermie. Ce mécanisme, nécessitant la formation du complexe BAG3-HspB8, augmente la survie des cellules probablement par l’élimination des protéines agrégées générées au cours du stress thermique / The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. Thus, we aimed to analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent cells. We demonstrated that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases the survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery leading to an increased level of necrotic cell death in NFκB incompetent cells. Moreover, when autophagy is artificially induced in these cells, the heat shock cytotoxicity is turned back to normal. We showed that the key regulators of autophagy (mTOR complex, and class III PI3Kinase complex) are not regulated by NFκB after heat shock. In contrast, we observed that aberrantly folded/aggregated proteins accumulation is a prime event in the activation of NFκB -mediated autophagy. Moreover, NFκB -depleted cells accumulate higher levels of protein aggregates induced by either heat shock treatment or mutated form of HspB5, indicating that the protein quality control process seem to be altered in these cells. This alteration could be caused by a defect in BAG3-HspB8 complex formation in NFκB -depleted cells. We demonstrated that heat shock treatment induces a NFB-dependent overexpression of the bag3 and hspb8 genes. Moreover, the accumulation of BAG3-HspB8 complex in heat shocked NFκB -competent cells is inhibited by NFκB depletion. Our findings how / prove / highlight revealed that NFκB -induced autophagy during heat shock recovery is an additional response to protein denaturation/aggregation induced by heat shock. This process depends on the BAG3-HspB8 complex formation and increases cell survival, probably through clearance of aggregated proteins

Les signatures histopathologiques de principales maladies neurodégénératives - maladie d'Alzheimer et la maladie de Parkinson - sont les enchevêtrements neurofibrillaires formés par la protéine tau et les corps de Lewy, formés par l'α-synucleine agrégée. Les mécanismes précis du repliement et de l'agrégation de ces protéines, pour la protéine tau comme pour l'α-synucleine, ne sont pas totalement compris à ce jour. Ici, nous nous sommes intéressés à cette question en utilisant des modèles in vitro et in vivo. En étudiant l'agrégation tau in vitro, nous avons mis en évidence un nouvel auto- assemblage réversible de tau, qui dépend de la température et de la présence d’ions zinc, et qui est a priori différent de l'agrégation de tau en présence d'inducteurs d'agrégation tels que l'héparine. Ce processus pourrait néanmoins être impliqué dans les premières étapes de l'agrégation pathologique de tau. Dans une deuxième partie nous avons développé des modèles murin pour étudier les dysfonctionnement de l’α-synucleine. Nous avons montré que l’α-synucleine est directement impliquée dans le développement embryonnaire de régions spécifiques du système nerveux, et qu'elle a des propriétés modulatrices seulement sur les neurones dopaminergiques de la substantia nigra, qui sont touchés dans la maladie de Parkinson.Les résultats obtenus dans nos études de deux protéines qui subissent une agrégation pathogène et forment des inclusions intracellulaires ont contribué à la compréhension des processus moléculaires et cellulaires associés à la dégénérescence neuronale, ce qui fournira de nouvelles pistes pour développer de nouvelles stratégies de thérapies de maladies neurodégénératives. / The histopathological hallmarks of the most common neurodegenerative diseases – Alzheimer’s disease and Parkinson’s disease are neurofibrillary tangles formed by tau protein and Lewy bodies inclusions formed by aggregated α-synuclein. The formation and accumulation of these proteins into inclusions cause functional disruptions of the cytoskeleton and leads to neuronal degeneration. The precise mechanisms of tau and synuclein misfolding and aggregation leading to those cellulare incluses, even though very studied, are not fully understood neither for tau protein nor for α-synuclein.Here we have addressed this question using both in vitro and in vivo models. Investigating tau aggregation in vitro, we have found a reversible self-assembly of tau, which depends on temperature and is induced by zinc ions, which is different from the tau aggregation in the presence of aggregation-inducers such as heparin. This process could be implicated in the first steps of tau pathological aggregation. In a second part, we have developed a mouse model for studying the α-synuclein dysfunction. We have shown that α- synuclein is directly involved in the embryonic development of the specific regions of the nervous system, and that it has modulating effect only on the populations of dopaminergic neurons of substantia nigra, which are affected in Parkinson’s disease.Results obtained in our studies of two proteins that undergo pathogenic aggregation and form intracellular inclusions contributed to understanding of molecular and cellular processes associated with neuronal degeneration, which is important for the development of new disease-modifying therapies of neurodegenerative disorders.

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

In this thesis we implement an ensemble of sequence analysis strategies aimed at identifying functional and structural protein features. The first part of this work was dedicated to two case studies of specific proteins analyzed to provide candidate functional positions for experimental validation: the protein alpha-synuclein (αsyn) and the alanine racemases protein family. In the case of αsyn, the objective was to predict its aggregation prone regions. For the alanine racemase protein family, the scope was to predict sites responsible for substrate specificity. In these two studies, computational predictions allowed systematically exploring potentially functionally relevant protein sites in an efficient manner that may not be possible to implement with traditional experimental approaches. Our strategy provided a powerful forecasting tool for the selection of candidate sites to be later verified experimentally. In the second part, we analyze the role of intrinsic disorder (ID) as a modulator of protein function in different organisms and cellular processes, which is largely unexplored. As key components of the diverse cellular pathways, disordered proteins are often involved in many diseases, including cancer and neurodegenerative diseases. Thus, there is an impeding need to unveil the general principles underlying the role of ID in proteins. We provide a multi-scale analysis of the involvement of ID in protein function starting with a large-scale analysis at genomic level of the role of ID in Arabidopsis, zooming in into the specific processes of vesicular trafficking in Human and yeast, and finally focusing on specific proteins of diverse organisms. The results of this thesis provide a better understanding of the functional roles mediated by ID in different organisms and biological processes, such as acting as flexible linkers connecting structured domains, mediating protein-protein interactions, and assisting the quick assembly of large macromolecular complexes. In addition, we present evidence of the use of ID as a mechanism to increase the complexity of protein and biological networks, and as a means to increase the adaptability of proteins in specific processes. Thus, our results contribute to elucidating the relationship between network and organismal complexity and ID, while they also provide evidence of the evolutionary advantages offered by ID.