Dissertations / Theses on the topic 'Bacterial Small Heat Shock Protein'
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Studer, Sonja. "Chaperone activity and oligomerization of bacterial small heat shock proteins /." [S.l.] : [s.n.], 2002. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=14550.
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Collier, Miranda. "Small heat shock protein interactions with in vivo partners." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:24cf8041-c82d-4bc4-87a7-0ae7e38f1879.
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Small heat-shock proteins (sHsps) are part of a broad cellular sys- tem that functions to maintain a stable proteome under stress. They also perform a variety of regulatory roles at physiological conditions. Despite the multitude of sHsp targets, their interactions with partners are not well understood due to highly dynamical structures. In this thesis, I apply a variety of biophysical and structural approaches to examine distinct interactions made by the abundant human sHsps αβ-crystallin and Hsp27. First, I find that αβ-crystallin binds a cardiac-specific domain of the muscle sarcomere protein titin. A cardiomyopathy-causative variant of αβ-crystallin is shown to disrupt this interaction, with demonstrated implications for tissue biomechanics. Next, I investigate the conformation and unfolding behaviour of another sarcomere-associated protein, filamin C, finding support for the hypothesis that it is mechanosensitive. This leads into an interrogation of the interaction between filamin C and Hsp27, which we find is modulated by phosphorylation of Hsp27. This modulation only manifests during filamin C unfolding, pointing toward a protective chaperoning mode against over-extension during mechanical stress. This finding is bolstered by up-regulation and interaction of both proteins in a mouse model of heart failure. I establish a system for similar studies of a third sHsp, cvHsp, which is muscle-specific and implicated in various myopathies but scantly understood at the molecular level compared to αβ-crystallin and Hsp27. Finally, I probe the stoichiometries and kinetics of complexes formed between αβ-crystallin and Hsp27 themselves, which co-assemble into a highly polydisperse ensemble. This involved the development of a high-resolution native mass spectrometry method for disentangling heterogeneous systems. Together these findings add to our understanding of the roles and mechanisms of ATP-independent molecular chaperones.
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Franzmann, Titus Marcellus. "Chaperone mechanism of the small heat shock protein Hsp26." kostenfrei, 2008. http://mediatum2.ub.tum.de/doc/652224/652224.pdf.
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Sund, Derrick T. "Replica Exchange Molecular Dynamics of a Small Heat Shock Protein." Thesis, The University of Arizona, 2011. http://hdl.handle.net/10150/144990.
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Morris, Amie Michelle. "Structure and function of the mammalian small heat shock protein Hsp25." Access electronically Access electronically, 2007. http://www.library.uow.edu.au/adt-NWU/public/adt-NWU20080605.104334/index.html.
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Carson, Kenneth Harris. "Study and characterization of a novel small heat shock protein from Babesia." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1813.
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di, Bard Barbara Lelj Garolla. "Self-association and chaperon activity of the small heat shock protein 27." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31382.
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Human Hsp27 is a member of the small heat shock protein family that is over-expressed during cellular stress and that is involved in biological functions ranging from inhibition of apoptosis to regulation of cellular glutathione levels. In addition, Hsp27 is an ATP-independent molecular chaperon that binds to unfolding peptides and inhibits their precipitation. Roles for Hsp27 in several human diseases have also been proposed. For example, the expression of Hsp27 by several human tumors has been noted as a potential diagnostic feature or a therapeutic target. Increasing evidence indicates that the biological functions of Hsp27 are linked to the reversible self-association of the protein to form large oligomers in a process that is at least in part regulated by reversible phosphorylation of three Ser residues. The three-dimensional structure of Hsp27 is not available, and relatively few rigorous physical studies of the protein have been reported. In the present study, analytical ultracentrifugation has been used to define self-association of Hsp27 and selected variants as a function of protein concentration, pH, temperature, and ionic strength to evaluate the role of structural domains believed to be functionally significant. These results are correlated with the chaperon activity, as determined by monitoring the inhibition of insulin unfolding, and with the kinetics of subunit exchange, monitored by fluorescence resonance energy transfer. The results establish that wild-type Hsp27 forms a distribution of oligomers that ranges from dimers to at least 32-mers and that oligomerization is highly regulated by temperature but not ionic strength or pH. Moreover, the oligomeric size of Hsp27 increases with increased temperature in a manner that correlates well with increased chaperon activity and rate of subunit exchange. Comparison of results from all three types of experiments obtained for the wild-type protein to those obtained with Hsp27 variants has led to the development of a model for Hsp27 self-association and chaperon activity.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Dabbaghizadeh, Afrooz. "Structure and function of mitochondrial small heat shock protein 22 in Drosophila melanogaster." Doctoral thesis, Université Laval, 2018. http://hdl.handle.net/20.500.11794/34491.
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Les petites protéines de choc thermique (sHsps) ont été découvertes initialement chez Drosophila. Les membres de cette famille sont des chaperons moléculaires sont présentsdans la plupart des organismes eucaryotes et procaryotes et certains virus. En plus d’être induites en réponse à la plupart des stresseurs dont un choc thermique, elles sont également exprimés en absence de stress. Les sHsps forment des structures dynamiques s'assemblant en oligomères et elles sont essentielles durant les conditions de stress en empêchant l'agrégation des protéines dénaturées et en favorisant leur repliement par des chaperons moléculaires dépendants de l'ATP. Le génome de Drosophila melanogastercode pour 12 sHsp, qui ont des profils d'expression développementaux, des localisations intracellulaires diverses et des spécificités de substrats distincts. DmHsp22 est jusqu'à présent la seule sHsp localisée dans les mitochondries avant et après un choc thermique. Elle est préférentiellement régulée lors du vieillissement et en réponse à la chaleur et aux stress oxydants. La surexpression de DmHsp22 augmente la durée de vie et la résistance au stress et sa régulation négative est préjudiciable. C'est un chaperon efficace, qui pourrait être impliqué dans la réponse mitochondriale au dépliement protéique (UPRMT). Cependant, le mécanisme exact de son action est mal compris. Structurellement, DmHsp22 forme une population d'oligomères semblable aux nombreux sHsps de métazoaires et différente deDmHsp27. L'alignement des séquences de la région ACDde DmHsp22 avec des sHsp de drosophile et d'autres organismes a démontré la présence de trois résidus d'arginine hautement conservés dans ce domaine. Une forte conservationde ces résidus suggère leur implication possible dans la structure et la fonction de DmHsp22. La substitution des résidus d'arginine hautement conservés dans les sHsps de mammifères est associée à certaines pathogenèses et déclenche des changements de conformation des protéines ainsi que l'agrégation des protéines intracellulaires. La mutation de l'arginine en glycine au niveau de trois résidus hautement conservés d'ACD dans DmHsp22 (R105, R109, R110) résulte en une population oligomérique qui, dans le cas de R110G, perturbe la structure et provoque la formation de petits oligomères. Bien que DmHsp22 ainsi que les mutants aient été caractérisés comme des chaperons efficaces in vitro, les mécanismes d'action exacts dans les mitochondries et l'information sur le comportement protecteur nécessitent la détermination du réseau d’interaction in vivo. Nous avons utilisé la technique capture d’immunoaffinité (CIA) pour récupérer 60 protéines qui interagissent spécifiquement avec DmHsp22 in vivo pendant le traitement normal et thermique, dans le surnageant des cellules de mammifères exprimant la DmHsp22. L’CIA effectuée sur la fraction mitochondriale a permis d’identifies 39 protéines qui interagissent spécifiquement avec DmHsp22. La combinaison de l’IAC avec l'analyse par spectroscopie de masse de mitochondries de cellules HeLa transfectées avec DmHsp22 a conduit à l'identification de partenaires de liaison à DmHsp22 dans des conditions de normales et de choc thermique. L'interaction entre DmHsp22 et deux autres chaperons mitochondriaux a été validée par immunobuvardage. Notre approche a montré que les cellules HeLa exprimant DmHsp22 augmentent la consommation d'oxygène mitochondrial et les teneurs en ATP, ce qui confère un nouveau rôle à DmHsp22 dans les mitochondries. En outre, l'activité d’une luciférase exogène a légèrement augmenté dans les cellules HeLa exprimant DmHsp22 après que l'activité enzymatique ait été réduite à la suite de l'exposition à la chaleur. En résumé, ce projet a permis de caractériser la structure oligomérique de DmHsp22 et un certain nombre de mutants dans le domaine alpha cristallin tout en fournissant un rôle potentiel mécanistique dans l’homéostase mitochondriale. La détermination du réseau mitochondrial de DmHsp22 suggère son importance dans cette organelle non seulement en tant que chaperon moléculaire, mais aussi en tant que protéine impliquée dans plusieurs fonctions cellulaires significatives.The small heat shock proteins (sHsps) were first discovered in Drosophila. Members of this family are molecular chaperones and are present in most eukaryotic and prokaryotic. Although, they are induced in response to most of the stressors including heat shock, they are also expressed in absence of stress. SHsps for mdynamic structures that assemble into oligomers which are essential during stress conditions by preventing aggregation of denatured proteins and promoting their folding by ATP dependent molecular chaperones. Drosophila melanogaster genome encodes 12 sHsps, that have developmental expression patterns, diverse intracellular localizations and distinct substrate specificities. DmHsp22 is up to now the only sHsp localized in mitochondria before and after heat shock. It is preferentially regulated during ageing and in response to heat and oxidative stresses. Over-expression of DmHsp22 increases lifespan and resistance to stress and its down-regulation is detrimental. It is an efficient chaperone and could be involved in the mitochondrial unfolding protein response (UPRMT). However, the exact mechanism of its action is poorly understood. Structurally, DmHsp22 forms one population of oligomers similar to the many metazoan sHsps but DmHsp27. Sequence alignment of DmHsp22 with sHsps in Drosophilaand other organisms at the alpha crystalline domain (ACD) region demonstrated the presence of three highly conserved arginine residues in this domain. Strong conservation of these residues suggest their possible involvement in structure and function of DmHsp22. Substitution of highly conserved arginine residues in mammalian sHsps is associated with some pathogenesis and triggers protein conformational changes as well as intracellular protein aggregation. Mutation of arginine to glycine at three highly conserved residues of ACD in DmHsp22 (R105, R109, R110) results in one oligomeric population as well which in the case of R110G disrupts the structure and causes formation of smaller oligomers. Although DmHsp22 as well as mutants have been characterized as effective in vitro chaperones, the exact mechanism(s) of action in mitochondria and information about protective behavior requires defining of in vivoprotein interacting network. We have used immunoaffinity conjugation (IAC) technique to recover 60 proteins that specifically interact with DmHsp22 in vivo during normal and heat treatment using cell extract of mammalian cells expressing DmHsp22. The IAC performed on mitochondrial fraction identified 39 proteins that specifically interact with DmHsp22. Combination of IAC with mass spectroscopy analysis of mitochondria of HeLa cells transfected with DmHsp22 resulted in identification of DmHsp22-binding partners under normal andunder heat shock conditions. Interaction between DmHsp22 and two other mitochondrial chaperones was validated by immunoblotting. Our approach showed that HeLa cells expressing DmHsp22 increase maximal mitochondrial oxygen consumption and ATP contents which provides a new mechanistic role for DmHsp22 in mitochondria. Further more, exogenous luciferase activity slightly increased in HeLa cells expressing DmHsp22 after the enzyme activity reduced as a result of exposure to heat. In summary, this project has characterized the oligomeric structure of DmHsp22 and a number of mutants inthe alpha crystalline domain while providing a potential mechanistic role in mitochondrial homeostasis. Determining mitochondrial network of DmHsp22 suggest its importance in this organelle not only as a molecular chaperone but also as a protein involved in several significant cellular functions.
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Friedrich, Kenneth Lane. "Dynamic behavior of small heat shock protein subunits and their interactions with substrates." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280410.
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Small heat shock proteins (sHsps) are oligomeric proteins expressed by cells in response to high temperatures. It is believed that sHsps are produced as a defensive mechanism against temperature stress and act as molecular chaperones by binding and protecting heat-labile proteins from irreversible aggregation. Binding results in the formation of sHsp/substrate complexes from which substrate can later be refolded by ATP-dependent chaperones. Despite past investigations, many aspects of this model remain poorly defined. Results presented here provide new insight into the mechanism of sHsp action. sHsp chaperone activity and sHsp oligomerization are closely linked. Therefore, an understanding of the oligomeric structure, subunit number, and subunit dynamics is essential to understanding sHsp action. Three sHsps were analyzed for these properties: PsHsp18.1 from pea, TaHsp16.9 from wheat, and SynHsp16.6, from the cyanobacterium Synechocystis. In solution, SynHsp16.6 is a duodecamer, while TaHsp16.9 and PsHsp18.1 are dodecamers. An equilibrium between an oligomeric and suboligomeric state was observed for PsHsp18.1 and SynHsp16.6. Increasing temperatures resulted in the reversible dissociation of the TaHsp16.9 oligomer into a suboligomeric species. These results indicate that subunit dynamics are important for sHsp function. Interactions between sHsp and substrate in sHsp/substrate complexes and the mechanism by which substrate is transferred to refolding chaperones are poorly defined. C-terminal affinity-tagged sHsps were used to investigate these issues. This analysis revealed that while some sHsp subunits within sHsp/substrate complexes remain dynamic, complex size remains unchanged and association of substrate with sHsp is not similarly dynamic. These data suggest a model in which ATP-dependent chaperones associate directly with sHsp-bound substrate to initiate refolding. The homologous TaHsp16.9 and PsHsp18.1 are structurally similar. However, TaHsp16.9 interacts differently with substrate and is less effective at protecting substrate than PsHsp18.1. Studies with chimeric sHsps made between PsHsp18.1 and TaHsp16.9 revealed that the N-terminal arm is involved in subunit affinity, substrate protection, and substrate refolding, but interactions between the N-terminal arm and C-terminal domain are also critical for these aspects of chaperone activity. Additionally, the first ten residues of the N-terminal arm play a role in sHsp subunit affinity and substrate protection, but are unimportant for substrate protection.
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Muchowski, Paul J. "Structural and functional characterization of human alphaB-crystallin, a small heat-shock protein and molecular chaperone /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/5676.
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Bentley, Nicola Jane. "Structural and biochemical analysis of a small heat shock protein, Hsp26, from Saccharomyces cerevisiae." Thesis, University of Kent, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304620.
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Iburg, Manuel. "Non-canonical small heat shock protein activity in health and disease of C. elegans." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/22439.
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Die erfolgreiche Synthese und Faltung von Proteinen ist eine Voraussetzung der Zellfunktion und ein Versagen der Proteinhomöostase führt zu Krankheit oder Tod. In der Zelle sichern molekulare Chaperone die korrekte Faltung der Proteine oder tragen zur Entsorgung unwiederbringlich fehlgefalteter Proteinsubstrate bei. Unter diesen Chaperonen sind kleine Hitzeschockproteine (sHsp) ein ATP-unabhängiger Teil des Proteostasenetzwerks.
In dieser Arbeit habe ich das bisher wenig erforschte sHsp HSP-17 aus C. elegans untersucht. Im Gegensatz zu anderen sHsps zeigte HSP-17 nur eine geringe Aktivität beim Verhindern der Aggregation von Proteinsubstraten. Stattdessen konnte ich in vitro zeigen, dass HSP-17 die Aggregation von Modellsubstraten fördert, was hier für Metazoen-sHsps erstmals gezeigt wurde. HSP-17 kopräzipitiert mit Substraten und modifiziert deren Aggregate möglicherweise. HSP-17 kolokalisiert in vivo mit Aggregaten, und seine aggregationsfördernde Aktivität konnte ich für das physiologische Substrat KIN-19 und heterolog exprimierte polyQ-Peptide validieren. Durch ex vivo Analysen konnte ich zeigen, dass die Aktivität von HSP-17 für die Fitness relevant ist
In einem zweiten Projekt habe ich zur Entwicklung eines neuen Modelles für Aß-Pathologie in C. elegans beigetragen, welches substöchiometrische Markierungen verwendet, um eine zeitnahe Visualisierung der Aß-Aggregation in spezifischen Zelltypen zu ermöglichen. Das Modell spiegelt bekannte Phänotypen der Aß-Proteotoxizität aus Menschen und bestehenden C. elegans Aß-Stämmen wider. Interessanterweise zeigt eine Untergruppe der Neuronen, die IL2-Neuronen, eine höhere Anfälligkeit für die Aggregation und Proteotoxizität von Aß1-42. Eine gezielte Reduktion von Aß1-42 in IL2 Neuronen führt zu einer systemischen Reduktion der Pathologie. Somit bietet das Modell eine neue Plattform, um die Bedeutung molekularer Chaperone, wie z. B. der sHsps, für Amyloidosen zu untersuchen, auch im Hinblick auf menschliche Erkrankungen.Successful synthesis and folding of proteins is a prerequisite for cellular function and failure of protein homeostasis leads to disease or death. Within the cell, molecular chaperones ensure correct protein folding or aid in the disposal of terminally misfolded protein substrates. Among these chaperones, small heat shock proteins (sHsps) are ATP-independent members of the proteostasis network. In this work, I analyzed the so far under-researched C. elegans sHsp HSP-17. Unlike other sHsps, HSP-17 exhibited only weak activity in preventing aggregation of protein substrates. Instead, I could show in vitro that HSP-17 can promote the aggregation of protein substrates, which is the first demonstration for metazoan sHsps. HSP-17 co-precipitates with substrates and potentially modifies the aggregates. HSP-17 colocalizes with aggregates and pro-aggregation activity is present in vivo, which I demonstrated for the physiological substrate KIN-19 and heterologously expressed amyloidogenic polyQ peptides. By physiological, biochemical and proteomic analysis I showed that HSP-17 activity is relevant for organismal fitness In a second project, I contributed to the development and characterization of a novel model of Aß pathology in C. elegans. This new AD model employs sub-stoichiometric labeling to allow live visualization of Aß aggregation in distinct cell types. The model mirrors known phenotypes of Aß proteotoxicity in humans and existing C. elegans Aß strains. Interestingly, a subset of neurons, the IL2 neurons, is shown to be more vulnerable to Aß proteotoxicity and targeted depletion of Aß in these neurons systemically ameliorates pathology. Thereby, the model presents a new platform to assess the relevance of molecular chaperones such as sHsps in amyloidosis with a perspective on human disease.
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Münkemer, Lea Johanna [Verfasser]. "alphaB-Crystallin: Insights into a small heat shock protein with NMR-spetroscopy / Lea Johanna Münkemer." Berlin : Freie Universität Berlin, 2019. http://d-nb.info/1192304187/34.
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Jaya, Nomalie Naomi. "SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/193550.
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sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.
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Ustyugov, Alexey. "Expression and function of the small heat shock protein Hsp27 during embryogenesis of zebrafish Danio rerio." Online access for everyone, 2007. http://www.dissertations.wsu.edu/Thesis/Fall2007/a_ustyugov_080307.pdf.
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Crack, Julie. "Functional analysis of domains within p26, a small heat shock/Ã-crystallin protein from Artemia franciscana." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0017/MQ57251.pdf.
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Iburg, Manuel [Verfasser]. "Non-canonical small heat shock protein activity in health and disease of C. elegans / Manuel Iburg." Berlin : Humboldt-Universität zu Berlin, 2021. http://d-nb.info/1227925131/34.
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Morrison, Lisa E. "A cardioprotective role for the small heat shock protein, alpha B-crystallin, in ischemia-reperfusion injury /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2003. http://wwwlib.umi.com/cr/ucsd/fullcit?p3112973.
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Lee, Sung Mun. "Role of aggregation conditions and presence of small heat shock proteins on abeta structure, stability and toxicity." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/3905.
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AlzheimerÂs disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In AlzheimerÂs disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.
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Ohan, Nicholas. "An examination of small heat shock protein gene expression in Xenopus laevis embryos and A6 kidney epithelial cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq21373.pdf.
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Paul, Megan Rose. "Arabidopsis thaliana eukaryotic elongation factors eEF1B-a(1 and 2), eEF1b-B(1 and 2) and their relationship to the plant heat shock response/small heat shock protein system." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192561.
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Kaur, Paramjit. "Interleukin 1 (IL-1) and Tumour Necrosis Factor (TNF) increase the phosphorylation of the small heat shock protein, HSP27." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359810.
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Kinzel, Linda. "Targeting the heat shock protein 90 with the novel small-molecule inhibitor NW457 sensitizes tumor cells to ionizing radiation." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-177602.
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Bissonnette, Sarah Ayano. "Degradation of the E. coli small heat-shock proteins by the AAA+ protease lon : significance to protein quality-control." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/58167.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010."February 2010." Cataloged from PDF version of thesis.
Includes bibliographical references (p. 118-127).
The refolding and elimination of damaged and aggregated proteins requires the concerted effort of several branches of the protein quality-control network. This network includes refolding chaperones, disaggregases, holdases and proteases. Many years of investigation have led to a partial understanding of how different branches of the protein quality-control network cooperate with each other to accomplish the critical task of refolding or eliminating damaged and aggregated proteins. Here we investigate cooperation between the Lon protease and the IbpA and lbpB small heat-shock protein (sHSP) holdases in the model organism, Escherichia coli. sHSPs are molecular chaperones that bind unfolded proteins and prevent their irreversible aggregation. sHSPs contain a central a-crystallin domain flanked by variable N- and C-terminal tails. These tails are responsible for the higher-order oligomerization, and therefore the chaperone functions, of sHSPs. The E. coli genome contains two sHSPs, ibpA and ibpB. We find that IbpA and lbpB are substrates of the Lon protease when in their free form, and also when they are bound to unfolded client proteins in vivo and in vitro. Interestingly, unlike other known substrates of AAA+ proteases, lbpA and lbpB seem to be recognized through a structural feature of their conserved a-crystallin domain, rather than through peptide motifs near their N- or C-termini.
(cont.) Furthermore, we find that IbpB facilitates the degradation of lbpA both in vivo and in vitro, and that the mechanism by which IbpB stimulates IbpA degradation is most likely through directly interacting with lbpA and making IbpA a better substrate, rather than by activating Lon and making Lon better able to degrade IbpA. Finally we investigate the importance of the degradation of lbps that are bound to aggregated client proteins and find that degradation of client-bound Ibps by Lon facilitates the refolding of lbp-bound clients. These data therefore uncover a previously undescribed connection between the proteolytic branch and the holdase branch of the protein quality-control network. Furthermore, this work demonstrates that in addition to being important for the degradation of damaged or misfolded proteins, proteolysis also has a novel role in the refolding of aggregated proteins.
by Sarah Ayano Bissonnette.
Ph.D.
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Hantke, Ingo [Verfasser]. "Protein quality control and antibiotics: the role of the small heat shock protein YocM and the disaggregase ClpC in B. subtilis / Ingo Hantke." Hannover : Gottfried Wilhelm Leibniz Universität, 2019. http://d-nb.info/1197227415/34.
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Li, Song. "Shear stress-induced signaling in vascular endothelial cells : roles of focal adhesion kinase, small GTPases and heat shock protein 27 /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9808991.
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Qian, Jiang. "The Role of Small Heat Shock Protein 20 and Its Phosphorylation in the Regulation of Cardiac Function and Ischemia/Reperfusion Injury." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1273168811.
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Kwan, Wai-yin, and 關偉然. "Glucose-regulated protein 78 as a novel target of BRCA1 for inhibitingstress-induced apoptosis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B4163391X.
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Kwan, Wai-yin. "Glucose-regulated protein 78 as a novel target of BRCA1 for inhibiting stress-induced apoptosis." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B4163391X.
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Le, Thanh Ha. "Optimisation of active recombinant protein production, exploring the impact of small heat-shock proteins of Escherichia coli, IbpA and IbpB, on in vivo reactivation of inclusion bodies." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975691554.
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Hanazono, Yuya. "Structural studies on the mechanism of protein folding." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/188506.
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Aatre, Rajani D. "Developmental changes in isoform composition and multimer formation of a small heat shock/Ã-crystallin protein in the brine shrimp (Artemia franciscana)." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ36385.pdf.
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Kröber-Boncardo, Constanze [Verfasser]. "New Insights into Leishmania Stress Tolerance: The Impact of the Small Heat Shock Protein 23 and Casein Kinase 1.2 / Constanze Kröber-Boncardo." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1229625666/34.
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Cockburn, Ingrid Louise. "Modulation of Plasmodium falciparum chaperones PfHsp70-1 and PfHsp70-x by small molecules." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1001747.
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The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
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Acosta-Sampson, Ligia I. "In vitro interactions of the small heat shock protein chaperone human [alpha]B-crystallin with its physiological substrates in the lens [gamma]-crystallins." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/61787.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
In title on title-page "[alpha]" and "[gamma]" appear as lower case Greek letters. Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (p. 160-175).
The passive chaperone a-crystallin, a small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the [beta][gamma]-crystallins. It is composed of two subunits (~ 20 kDa) aA- and [alpha]B-crystallin (aA- and [alpha]B-Crys), which form a hetero-oligomeric, polydisperse complex of ~ 800 kDa in the lens. Aggregates isolated from mature-onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of [beta][gamma]-crystallins, as well as a-crystallins. I have studied the chaperone function of human [alpha]B-crystallin interacting with its physiological human [gamma]-crystallin substrates. Human [gamma]D-crystallin ([gamma]D-Crys) and [gamma]C-crystallin ([gamma]C-Crys) are st[alpha]Ble and long-lived mammalian [gamma]-crystallins localized in the lens nucleus. Human [gamma]S-crystallin ([gamma]S-Crys) is [alpha]Bundant in the lens outer cortex. All three [gamma]-crystallins can refold in vitro to their native state after unfolding in high concentrations of guanidine hydrochloride (GdnHCl). However, in buffer or very low denaturant concentrations (< 1 M GdnHCl) aggregation of refolding [gamma]-crystallin intermediates competes with productive refolding. Diluting unfolded [gamma]C-, [gamma]D-, or [gamma]S-Crys to low GdnHCl concentrations (at 100 [mu]g/ml, 37°C) resulted in the protein population partitioning between productive refolding and aggregation pathways. [gamma]D-, [gamma]C- or [gamma]S-Crys protein was allowed to refold and aggregate in the presence of [alpha]B-Crys homo-oligomers at different mass-based ratios of [gamma]-Crys to [alpha]B-Crys. [gamma]D- and [gamma]C-Crys aggregation was suppressed to similar levels, whereas [gamma]S-Crys aggregation was not suppressed as strongly in assays measuring solution turbidity at 350 nm. SEC chromatograms of the products of suppression reactions showed the presence of a high molecular weight chaperone-substrate complex. This complex was still present 4 days after the suppression reaction was initiated. Experiments were performed with the [alpha]B-Crys chaperone added 2, 6, or 10 s, after dilution of unfolded [gamma]D-Crys out of high concentrations of denaturant. The results from these experiments showed that the partially folded, aggregation-prone species that is recognized by [alpha]B-Crys chaperone is populated within the first 10 s after refolding and aggregation were initiated. This time period coincided with the refolding of the C-terminal domain of [gamma]D-Crys as determined from kinetic refolding experiments in vitro. Human [gamma]D-Crys contains four Trp residues with one residue located in each quadrant of the protein. Intrinsic buried Trp fluorescence is quenched in the native state relative to the unfolded state of the protein due to intra-domain partial resonance energy transfer from the highly fluorescent Trp donors (W42 and W130) to the highly quenched acceptor Trps (W68 and W156). The efficient quenching of Trp68 and Trp156 depends on an unusual conformation of the Trp ring with respect to its backbone amide, as well as the presence of two tightly bound H2O molecules with oppositely oriented dipoles. Thus, intrinsic Trp fluorescence is a sensitive reporter of the protein conformation. Using a no-Trp mutant of [alpha]B-Crys (W9F/W60F), the conformation of the bound [gamma]D-Crys substrate in [gamma]D -- [alpha]B complexes was determined from intrinsic Trp fluorescence emission. The emission spectra for the substrate did not coincide with a native or fully unfolded conformation of the [gamma]D-Crys controls. To further characterize the conformation of each domain of [gamma]D-Crys in the substrate-chaperone complex, double-Trp [gamma]D-Crys mutants, which conserved the Trp pair in the N-terminal (W130F/W156F) or the C-terminal (W42F/W68F) domain, while the counterpart pair was changed to Phe, were used as substrates in aggregation suppression reactions. The fluorescence emission spectra for the double-Trp mutants in complex with Trp-less [alpha]B-Crys were similar and they did not coincide with the spectra for their respective native or unfolded double-Trp [gamma]D-Crys controls. These results indicated that the bound substrate remained in a partially folded state with neither domain native-like. Triple-Trp [gamma]D-mutants that conserved the highly fluorescent Trp residue in the N-terminal or C-terminal domains were also used as substrates in suppression of aggregation reactions with Trp-less [alpha]B-Crys chaperone. The fluorescence emission spectra of triple-Trp substrates in the substrate-chaperone complex indicated that these residues were not solvent exposed. These results suggest that Trp neighboring regions could be interacting directly with the [alpha]B-Crys chaperone. To further elucidate the specific region in the [gamma]-crystallins that interacts with [alpha]B-Crys in suppression assays, experiments were performed using single-domain constructs of [gamma]D-Crys. The isolated N-terminal ([gamma]D-Ntd) and C-terminal domains ([gamma]D-Ctd) of [gamma]D-Crys, expressed in E. coli, can refold to a native state upon dilution out of denaturant to low concentrations of GdnHCl. The C-terminal domain aggregated upon refolding out of high concentrations of denaturant, while the N-terminal did not under the same assay conditions. However, when [gamma]D-Ctd and [gamma]D-Ntd were unfolded and refolded together, [gamma]D-Ctd recruited [gamma]D-Ntd into the aggregate. [alpha]B-Crys suppressed the aggregation of the [gamma]D-Ctd and formed [gamma]D-Ctd -- [alpha]B complexes. Using W9F/W60F [alpha]B-Crys, I have determined, through the fluorescence emission of [gamma]D-Ctd tryptophans, that the [gamma]D-Ctd in the [gamma]D-Ctd --[alpha]B complexes was partially folded. Inhibition experiments in which the [gamma]D-Ntd and [gamma]D-Ctd isolated domains were refolded sequentially or simultaneously showed that [alpha]B-Crys preferentially recognized [gamma]D-Ctd. These in vitro results provide a model for how a-crystallin interacts with aggregation-prone substrates in vivo wherein an aggregation-prone region in the C-terminal domain of [gamma]D-Crys is exposed in the aggregation-prone species and this region is recognized by [alpha]B-Crys. These results also provide support for protein unfolding/protein aggregation models for cataract, with a-crystallin suppressing aggregation of damaged or unfolded proteins through early adulthood, but becoming saturated with advancing age.
by Ligia Acosta-Sampson.
Ph.D.
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Kinzel, Linda [Verfasser], and Anna [Akademischer Betreuer] Friedl. "Targeting the heat shock protein 90 with the novel small-molecule inhibitor NW457 aensitizes tumor cells to ionizing radiation / Linda Kinzel. Betreuer: Anna Friedl." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1064264980/34.
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Langston, Kelsey Murphey. "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/3822.
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Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.
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Peschek, Jirka [Verfasser], Johannes [Akademischer Betreuer] Buchner, Matthias [Akademischer Betreuer] Rief, Sevil [Akademischer Betreuer] Weinkauf, and Bernd [Akademischer Betreuer] Reif. "Structural and Functional Principles of the Small Heat Shock Protein alpha-Crystallin / Jirka Peschek. Gutachter: Matthias Rief ; Sevil Weinkauf ; Bernd Reif ; Johannes Buchner. Betreuer: Johannes Buchner." München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/104594548X/34.
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Zhang, Jie. "ROLE OF MEL-18 IN REGULATING PROTEIN SUMOYLATION AND IDENTIFICATION OF A NEW POLYMORPHISM IN BMI-1." UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_diss/685.
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Small ubiquitin-like modifier (SUMO) regulates numerous biological functions. In a previous study we found that sumoylation of HSF2 is involved in regulating HSF2 bookmarking function, but the mechanism that mediates this regulation was unknown. The results in my work support the intriguing hypothesis that polycomb protein, Mel-18, actually functions as an anti-SUMO E3 protein, interacting both with HSF2 and the SUMO E2 Ubc9, but acting to inhibit Ubc9 activity and thereby decrease sumoylation of the HSF2.
This study also suggested that Mel-18 negatively regulates the sumoylation of other cellular proteins, and we extend its targets to RanGAP1 protein. The results also show that RanGAP1 sumoylation is decreased during mitosis, and that this is associated with increased interaction between RanGAP1 and Mel-18. Previous studies showed little evidence of anti-SUMO E3 proteins, however, my study, taken together, found Mel-18 actually functions as a novel anti-SUMO E3 protein, interacting both with substrates and the SUMO E2 Ubc9 but acting to inhibit Ubc9 activity to decrease sumoylation of target proteins and also provide an explanation for how mitotic HSF2/RanGAP1 sumoylation is regulated. This finding also gives a clue for a future study direction in Mel-18 as a tumor suppressor: the anti-SUMO E3 function.
Additionally, we identify a single-nucleotide polymorphism in another human PcG protein, Bmi-1, that changes a cysteine residue within its RING domain, cysteine 18, to a tyrosine. This C18Y polymorphism is associated with a significant decrease in levels of the Bmi-1 protein. Furthermore, the C18Y Bmi-1 protein exhibits a very high level of ubiquitination compared to wild-type Bmi-1, suggesting that that the low levels of this form of Bmi-1 are due to its destruction by the ubiquitin-proteasome system. Consistent with this hypothesis, treatment of cells with the proteasome inhibitor MG-132 results in a significant increase in levels of C18Y Bmi-1. This is the first example of a polymorphism in human Bmi- 1 that reduces levels of this important protein.
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Blanchard, David Joseph. "Characterization of a Beta-glucosidase Aggregating Factor Responsible for the Null Beta-glucosidase Phenotype in Maize (Zea mays L.)." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/78093.
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β-Glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of aryl and alkyl β-D-glucosides as well as glucosides with a carbohydrate moiety such as cellobiose and other beta-linked oligosaccharides. In maize (Zea mays L.), β-glucosidase exists as 120 kD homodimers, but also forms high-molecular-weight (HMW) aggregates in certain maize inbreds (nulls). In this study we show that the null β-glucosidase phenotype is caused by the formation of HMW enzyme aggregates (>1.5 X 10⁶ Daltons), caused by a β-glucosidase aggregating factor (BGAF). BGAF is a 32 kD protein that binds specifically to β-glucosidase and renders it insoluble during extraction. The data unequivocally demonstrate that BGAF is solely responsible for β-glucosidase aggregation and insolubility, and thus, the apparent null phenotype. Additionally, I have isolated the cDNA encoding BGAF and have identified BGAF as a member of the small heat-shock protein (sHsp) family.
Interestingly, BGAF binds to both maize β-glucosidase isozymes (Glu1 and Glu2), but does not bind to their sorghum homolog Dhurrinase-1 (Dhr1; Sorghum beta-glucosidase), that shares 70% sequence identity with Glu1 and Glu2. Therefore, these proteins provide an excellent system to study functional differences at nonconserved residues and elucidate the mechanism of enzyme aggregation and insolubility. By examining the behavior of β-glucosidase chimeras in binding assays, I demonstrate that BGAF binding is conformation dependent, highly specific, and reminiscent of antigen-antibody interactions. Additionally, I have identified two disparate polypeptide segments in the primary structure of the maize beta-glucosidase isozyme Glu1 that form a BGAF binding site in the tertiary structure of the enzyme.Master of Science
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Fleckenstein, Tilly Thea [Verfasser], Johannes [Akademischer Betreuer] Buchner, and Michael [Akademischer Betreuer] Groll. "Sip1, a Unique Small Heat Shock Protein of the Nematode Caenorhabditis elegans : a Structural and Functional Characterization / Tilly Thea Fleckenstein. Betreuer: Johannes Buchner. Gutachter: Michael Groll ; Johannes Buchner." München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1079001840/34.
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Beck, Johannes Gerhard [Verfasser], Horst [Akademischer Betreuer] Kessler, and Iris [Akademischer Betreuer] Antes. "Structural Studies of N-methylated Cyclopeptides and of the Small Heat Shock Protein Hsp26 from S. cerevisiae / Johannes Gerhard Beck. Gutachter: Horst Kessler ; Iris Antes. Betreuer: Horst Kessler." München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/1024161714/34.
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Launay, Nathalie. "Voies de signalisation et rôle de l'αB-cristalline suite à la désorganisation du cytosquelette." Paris 6, 2006. http://www.theses.fr/2006PA066375.
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Le premier objectif de ce travail de recherche a été d’identifier les voies de signalisation impliquées dans la transduction du signal engendré par la perturbation/désorganisation spécifique des trois principales structures du cytosquelette au sein du système musculaire. Ces stress spécifiques se caractérisent par l’induction de la phosphorylation spécifique de l’B-cristalline, modification qui induit des changements de localisation et régulerait les fonctions de la protéine en augmentant son affinité pour certains éléments du cytosquelette. Dans un deuxième temps, le rôle de la phosphorylation de l’B-cristalline a été étudié dans le phénomène de résistance de cellules de carcinomes mammaires vis-à-vis des traitements anti-tumoraux altérant le cytosquelette. Lors de ce travail, nous avons mis en évidence que la phosphorylation du résidu sérine 59 de l’B-cristalline jouerait un rôle clé dans la régulation des propriétés anti-apoptotiques de la protéineThe first aim of this research was to identify the pathways involved in the signal transduction induced by specific cytoskeletal stresses in the muscular system. Cytoskeletal network disruption trigger the specific B-crystallin phosphorylation which may protect or stabilize the actin microfilaments and interfere with formation of IF aggregates during stresses exerted at the cytoskeleton. The second aim of this project was to study the role of phosphorylation in the regulation of the αB-crystallin anti-apoptotic function in chemotherapy resistance phenomenon in breast epithelial adenocarcinoma cells. We demonstrate that Ser59 phosphorylation of B-crystallin plays a crucial role in vinblastine-induced apoptosis in MCF7 cells, by down-regulating the anti-apoptotic function of B-crystallin. These results introduce the possibility of regulating the protective status of sHsp in cancer through modifying their state of phosphorylation
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Zhang, Xia. "Endothelial HSPA12B is a Novel Protein for the Preservation of Cardiovascular Function in Polymicrobial Sepsis via Exosome MiR-126." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etd/3129.
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Sepsis is the most frequent cause of mortality in most intensive care units. Cardiovascular dysfunction is a major complication associated with sepsis, with high mortality rates up to 70%. Currently, there is no effective treatment approach for sepsis.
The integrity of the endothelium is fundamental for the homeostasis of the cardiovascular system. Sepsis induces endothelial cell injury which is the key factor for multiple organ failure. The increased expression of adhesion molecules and chemokines in endothelial cell promotes leukocytes infiltration into the tissue. The loss of tight junction proteins and increased permeability of the endothelial cells will provoke tissue hypoxia and subsequent organ failure. Therefore, preservation of endothelial function is a critical approach for improving sepsis-induced outcome.
Here, we showed that endothelial specific protein HSPA12B plays a critical role in the preservation of cardiovascular function in polymicrobial sepsis. HSPA12B is the newest member of HSP70 family which predominantly expresses in endothelial cells. We observed that HSPA12B deficiency (HSPA12B-/-) exaggerated polymicrobial sepsis-induced endothelial dysfunction, leading to worse cardiac dysfunction. HSPA12B-/- significantly increases the expression of adhesion molecules, decreases tight junction protein levels and enhances vascular permeability. HSPA12B-/- alsomarkedly promotes the infiltration of inflammatory cells into the myocardium and inflammatory cytokine production.
We investigated the cardioprotective mechanisms of HSPA12B in sepsis induced cardiovascular dysfunction. Exosomes play a critical role in intercellular communication. Exosome is a natural vehicle of microRNAs. We found that exosomes isolated from HSPA12B-/- septic mice induced more expression of adhesion molecules in endothelial cells and inflammation in macrophages. Interestingly, the levels of miR-126 in serum exosomes isolated from HSPA12B-/- septic mice were significantly lowers than in WT septic mice. Importantly, delivery of miR-126 carried exosomes significantly improved cardiac function, suppressed the expression of adhesion molecules, reduced immune cell infiltration in the myocardium, and improved vascular permeability in HSPA12B-/- septic mice. The data suggests that HSPA12B is essential for endothelial function in sepsis and that miR-126 containing exosomes plays a critical role in cardiovascular-protective mechanisms of endothelial HSPA12B in polymicrobial sepsis.
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Salvà, Vila Lluís. "Les sHsps en surera: Estudis de funcionalitat." Doctoral thesis, Universitat de Girona, 2005. http://hdl.handle.net/10803/7619.
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Aquesta tesi es centra en la caracterització funcional d'una proteïna de xoc de calor de baix pes molecular (Small Heat Shock Protein - sHSP) de classe I de surera pel que fa a la seva capacitat per protegir les cèl·lules de l'estrès i per estabilitzar les membranes. Les sHsps són proteïnes que s'expressen en condicions d'estrès cel·lular. Encara que certs aspectes funcionals de les sHsps són ben coneguts, el nostre treball aporta informacions noves sobre el paper de les diferents regions de la proteïna, especialment de la regió N-terminal.L'objectiu concret d'aquest treball és determinar la funció termoprotectora de QsHsp17.4-CI, una sHsp de classe I oobtinguda a partir de les cèl·lules de fel·lema d'alzina surera, en un model bacterià i analitzar la importància de les diferents regions de la proteïna en aquesta funció. Amb aquesta finalitat s'han dissenyat dues proteïnes parcials derivades de QsHsp17.4-CI: una a la que li falta la regió N-terminal (C105) i una altra amb pràcticament tot el domini -cristal·lí deleccionat (N61), i una tercera, derivada de QsHs10-CI, a la que li falta la meitat del domini -cristal·lí (Hsp10). També s'estudia la possible capacitat estabilitzadora de membranes i la capacitat de modificar l'expressió d'altres Hsps quan s'expressa de forma heteròloga.
Els nostres resultats demostren que l'expressió de QsHsp17.4-CI protegeix a les cèl·lules d'E.coli de l'estrès tèrmic alhora que la regió N-terminal i la regió consens II del domini -cristal·lí són imprescindibles per aquesta funció de protecció.
En relació a un possible paper en les membranes, els estudis de localització subcel·lular mostren que QsHsp17.4-CI colocalitza amb la fracció membranes i que la regió N-terminal de la proteïna és responsable d'aquesta colocalització. No s'ha pogut demostrar, però, que la localització amb la membrana estigui associada a un efecte protector d'aquesta: en cap cas la sobrexpressió de les proteïnes modifica la composició d'àcids grassos i només N61, que no té acció termoprotectora, altera l'estat fisico-químic de la membrana.
En estudis d'expressió de novo en E.coli s'ha observat que, a diferència de les altres proteïnes heteròlogues, N61 activa l'expressió de la majoria de Hsps d'E.coli fent pensar en una possible relació entre l'estat físic de la membrana i l'activació de la resposta a l'estrès.
En resum, en aquest treball hem provat la capacitat protectora de QsHsp17.4 i aportem noves dades sobre la importància de la regió N-terminal i la regió consens II del domini -cristal·lí en aquesta funció. Per altra banda, es suggereix que QsHsp17.4 podria interaccionar amb la membrana d'E.coli i que la regió N-terminal seria imprescindible per aquesta interacció. Finalment hem determinat que les proteïnes que provoquen variacions en l'estat de fluïdesa de la membrana poden activar la resposta al xoc de calor per part de la cèl·lula bacteriana.
This thesis is focused in the functional studies of a Small Heat Shock Protein (sHsp). sHsps are expressed under stress conditions. Although some functional aspects of these proteins are known, our work aport new data about the role of the different protein regions, especially the N-terminal region.
The aim of this work is to demonstrate a thermotolerance effect of QsHsp17.4-CI in bacterial cells and to analyze the importance of the protein regions in this function. To achieve this objective two deletion mutants derived from QsHsp17.4-CI were designed: a protein lacking the N-terminal region (C105) and a protein where the entire -cristallin domain is missing (N61) and a third mutant, derived from QsHsp10-CI, that bears half of the -cristallin domain (Hsp10). To better understand the functional mechanism of sHsps we study the membrane stabilizing capacity of QsHsp17.4-CI as well as its capacity to modify other Hsps expression.
Our results demonstrate that the expression of QsHsp17.4-CI protects E.coli cells from a heat shock and that the N-terminal region and the consensus region II of the -cristallin domain are necessary for this protective function.
Related to a possible role in membranes, location studies suggest that QsHsp17.4-CI colocalizes with cell membrane fraction and that N-terminal region is important for this location. However, no relation between membrane localization and a protective effect has been demonstrated: Protein overexpression does not modify membrane fatty acid composition and only N61, which has no thermoprotection, changes membrane physical state.
Studies of E.coli de novo synthesis show that, unlike the other recombinant proteins, the overexpression of N61 activates the expression of almost all E.coli Hsps suggesting a possible relation between membrane physical state and the activation of the heat shock response.
As summary, in this work we have demonstrated the thermoprotective capacity of QsHsp17.4-CI and we contribute with new data about the importance of N-terminal region and consensus region II of -cristallin domain for this function. On the other hand, we suggest the possibility that QsHsp17.4-CI interacts with membrane and that N-terminal region is important for this interaction. Lastly, we have observed how changes in membranes fluidity state can activate heat shock response in bacterial cells.
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Mainz, Andi. "Beyond the limit." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16603.
Full textAbstract:
Strukturelle Untersuchungen mittels Lösungs-NMR Spektroskopie sind für supramolekulare Maschinen mit Molekulargewichten von mehr als 150 kDa nur beschränkt möglich. Die Festkörper-NMR mit Probenrotation im sogenannten magischen Winkel (MAS) stellt dagegen eine molekulargewichtsunabhängige Methode dar. Im Rahmen dieser Arbeit wurde eine neue Methode entwickelt, die die MAS NMR Spektroskopie an supramolekularen Komplexen in Lösung erlaubt. Proteinlösungen bilden demnach durch MAS und dessen Ultrazentrifugationseffekt homogene Proteinsedimente aus, in denen die rotatorische Diffusion großer Proteinkomplexe überwiegend aufgehoben ist. Auf diese Weise können klassische Festkörper-NMR Methoden angewandt werden, ohne dass Präzipitations- oder Kristallisationsverfahren erforderlich sind. In Kombination mit Proteindeuterierung, Protonendetektion sowie paramagnetischer Relaxationsverstärkung ermöglichte diese neuartige Methode die Zuordnung von Rückgrat-Amidresonanzen des 20S Proteasoms mit einem Molekulargewicht von 1,1 MDa. Weiterhin wurde diese Methode zur Untersuchung des kleinen Hitzeschockproteins alpha-B-Crystallin und dessen Cu(II)-Bindungseigenschaften genutzt. Das Chaperon (600 kDa) spielt eine wesentliche Rolle in der zellulären Proteinhomeostase. Verschiedenste NMR Techniken und andere biophysikalische Methoden zeigen, dass die konservierte alpha-Crystallin-Domäne ein Cu(II)-Ion nahe der Monomer-Monomer Interaktionsfläche mit pikomolarer Affinität bindet. Die Cu(II)-induzierte Freilegung von Substrat-Interaktionsflächen und Veränderungen in der dynamischen Quartärstruktur modulieren so die oligomere Architektur und die Chaperonaktivität von alpha-B-Crystallin. Die hier erstmals beschriebene MAS NMR Spektroskopie von sedimentierten Biomolekülen legt einen wichtigen Grundstein für zukünftige Struktur- und Dynamikuntersuchungen an großen molekularen Maschinen.Structural investigations of large biomolecules by solution-state NMR are challenging in case the molecular weight of the complex exceeds 150 kDa. Magic-angle-spinning (MAS) solid-state NMR is a powerful tool for the characterization of biomolecular systems irrespective of their molecular weight. In this work, an approach was developed, which enables the investigation of supramolecular modules by MAS NMR. Protein solutions can yield fairly homogeneous sediments due to the ultracentrifugal forces during MAS. Since rotational diffusion is impaired, typical solid-state NMR techniques can thus be applied without the need of precipitation or crystallization. This new approach in combination with protein deuteration, proton-detection and paramagnetic relaxation enhancement enabled the observation and the assignment of backbone amide resonances of a 20S proteasome assembly with a molecular weight of 1.1 MDa. Similarly, the approach was used to characterize the small heat-shock protein alpha-B-crystallin with respect to its Cu(II)-dependent chaperone activity. The chaperone (600 kDa) plays an essential role in cellular protein homeostasis. We show that the conserved alpha-crystallin core domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion near to the dimer interface with picomolar binding affinity. We suggest that Cu(II)-binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher-order assemblies of alpha-B-crystallin. In summary, MAS NMR employed to biomolecules in solution is a very promising tool to explore structural and dynamic properties of large biological machines with no upper size limit.
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Bhandari, Spraha. "Structural and Functional Characterisation of Small Heat Shock Protiens from Mycobacterium Marinum M." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4175.
Full textAbstract:
Small Heat Shock Proteins (sHSP) are ATP-independent molecular chaperones that exhibit diversity in structure, function and mode of action. They are present ubiquitously in all kingdoms of life. They function mainly by preventing the aggregation of non-native and destabilised proteins during stress as well as normal conditions. Their subunit molecular weight ranges from 12 kDa to 42 kDa and exist as higher order oligomers in their resting or native state. Structurally, these proteins have a tripartite domain organization with a structured α-crystallin domain (ACD) at the centre flanked by variable and flexible N-terminal domain and C-terminus. The protomers associate into dimers, which further assemble into higher order structures. The N-termini is longer than the C-termini, harbour sites for post-translational modifications, usually found buried within the oligomer and are required for substrate recognition and binding. The C-termini on the otherhand, harbour a conserved motif, called the I-X-I motif which facilitate formation of higher order structures. The thesis reports the structural and functional characterisation of three sHSPs (M1, M2 and M3) and their deletion constructs from Mycobacterium marinum M. In mycobacteria, these proteins are immunodominant antigens and are also used as biomarkers for disease identification. All the three proteins formed oligomers of different stoichiometry as determined through Size exclusion chromatography-Multi angle light scattering (SEC-MALS) experiments. Lysozyme aggregation assay was performed for assessing the ATP-independent molecular chaperone activity of these proteins. M1 and M3 were observed to be active while M2 was inactive. From the three sHSPs, one of the proteins, M3, crystallised and hence was taken up for structural investigations. The protein crystallised in different conditions and the structure was determined using data from a Se-Met derivative (Se-SAD) and molecular replacement (MR) phasing in space groups I23 (at 2.8 Å and 2.0 Å resolution, respectively) and C2221 (3.75 Å). The structure was a dodecamer with a cage-like architecture, exhibiting a 23 symmetry. The dodecameric assembly was observed to be hydrophobic inside and hydrophilic outside. The I-X-I mode of interaction, as observed in other sHSP structures, formed the dimer-dimer association and stabilized the cage. Electron micrographs collected for M3 protein, further confirmed that the structure reported was a dodecamer in solution. Further, on comparison of the high resolution sHSP structure of M3 with other reported structures, a similar arrangement of trimers was observed. This is the first report of a high resolution sHSP crystal structure from mycobacteria.
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Biswas, Sreeparna. "Small Heat Shock Proteins from Bacteria and a Bacteriophage." Thesis, 2020. https://etd.iisc.ac.in/handle/2005/4785.
Full textAbstract:
Proteins can unfold during heat and other types of stress and tend to aggregate with subsequent loss of function. This event is prevented by small Heat Shock Proteins (sHSPs) acting as molecular chaperones. sHSPs do not bind to native proteins but interact with unfolding proteins during stress conditions and transfer them to the other heat shock proteins for proper folding once normalcy is restored. sHSPs thus maintain cellular proteostasis and unlike the other heat shock proteins of higher molecular weight, sHSPs function in an ATP-independent manner. sHSPs are present in all kingdoms of life with many organisms having multiple sHSPs with little overall sequence identity.
The molecular weight of sHSPs varies between 12 to 40 kDa. The structure of -crystallin domain present in sHSPs is conserved and is flanked by flexible N- and C- terminal regions with roles mainly in substrate binding and oligomerization, respectively. sHSPs often form large oligomeric assemblies with the dimeric unit as the basic building block. sHSPs exist in a dynamic equilibrium by constant exchange of subunits between oligomers of various sizes. They have a broad substrate specificity, however, there is a dearth of knowledge on the exact mechanism of substrate binding and the necessity for the formation and exact functioning of different oligomers. The available structural information on sHSPs is limited as the three-dimensional structures of only a few higher order oligomers have been determined to date. To obtain insights into the functions and structures of sHSPs and the correlation between them and as a part of an ongoing project on sHSPs, we have carried out biophysical, biochemical and structural investigations on sHSPs of three bacteria: Bacillus cereus, Staphylococcus aureus and a cyanobacterium Synechococcus sp. WH7803; and a bacteriophage of Synechococcus sp. WH7803, called Synechococcus phage S-ShM2, which are presented in this thesis.
The full length and a few deletion constructs of these four sHSPs were generated. The chaperone activity of all the constructs was investigated using two denatured substrates: NdeI and lysozyme. The size and the content of the oligomers were determined by SEC-MALS and
DLS. The importance of the C-terminal I-X-I motif in higher oligomer formation and the role of the N-terminus in the chaperone activity and oligomerization of sHSPs were examined. Though crystallization of all the constructs was attempted, crystals of only the cyanophage sHSP (SM2), which diffracted to a low resolution of 7 Å could be obtained. SM2 crystallized as a 24-mer with 432 symmetry similar to the sHSP of Methanococcus jannaschii and one of the forms of AgsA of Salmonella typhimurium. The dimer is similar to that found in sHSPs of non-metazoans. Negative stain EM images showed that the protein is heterogeneous in nature with a major population of large cage-like assemblies and a minor population of smaller cages. 3D structures of both of them have been determined by Cryo-EM 3D image reconstruction at a resolution of ~ 8 Å. The larger particles are 60-mers with 52 symmetry and the smaller ones are 48-mers with 432 symmetry. The crystallographic and EM studies show that SM2 is capable of forming various types of oligomers, which vary in size and symmetry. This is the first structural report of a viral or a phage sHSP. This is the largest sHSP particle (60-mer) of all reported structures and the 52 symmetry is observed for the first time in sHSPs. Despite the variations, certain common features are observed in the three particles. Hexameric sub-assemblies made up of three dimers with 32 symmetry are present in all the three oligomers. Though the structures were determined at low resolutions, it is clear that the C-terminal I-X-I motif interacts with the neighboring molecules in a way similar to that observed in other sHSP structures. The flexibility of the C-terminal region combined with slight differences in the arrangement of dimers in the hexamer enabled the variation in oligomerization. Our studies confirmed the highly polydisperse and heterogenic nature of sHSPs, however, we could study the structures of three types of particles in isolation which revealed new features related to the oligomerization and symmetry of sHSPs.IISc fellowship
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Tsung-Yuan, Lee. "Regulation of small heat shock protein genes expression by heat shock factors transcriptome in Arabidopsis." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2301200621514900.
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Lee, Tsung-Yuan, and 李宗遠. "Regulation of small heat shock protein genes expression by heat shock factors transcriptome in Arabidopsis." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/47149842006878988298.
Full textAbstract:
碩士國立臺灣大學
植物科學研究所
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Heat shock (HS) response is a general physiological reaction to both prokaryotic and eukaryotic cells when temperature is suddenly elevated. The heat shock proteins (HSPs) induced by HS act as molecular chaperones to protect cells from HS damages. In plants, small HSPs (sHSP) represent the most abundant and complexes HSPs. And heat shock factors (HSFs) are known to involve in the regulatory mechanism of sHSP expression under HS stress. In Arabidopsis and rice, there are 21 and 25 HSFs, which are divided into three classes, respectively. That may implicate a complicated regulation mechanism (regulome) in higher plants. Other than heat shock, the regulation of sHSPs expression is still not clear. In this study, we studied the relationship between the sHSPs and HSFs expression under various stresses and to speculate the function of individual HSF. To investigate the details of the sHSPs regulome, we set up a negative selection system to screen mutants that can not respond to the HS-like induction. RT-PCR was used to analyze the HSF transcriptome to clarify the regulation of sHSP genes expression. We determined that all of sHSP genes were induced when seedlings were challenged with HS, amino acid analog (azetidine, Aze) and arsenite. Some of sHSPs, in response to cadmium and sodium nitroprusside (SNP, a NO donor), did not affect the expression of sHSPs. However, expression of some HSFs was not interfered by treatments. HsfA2a and three class B HSFs were induced by HS, Aze, and heavy metals treatments. The expression of HsfA7b was hyper inducted under HS than by other chemical inducers. One HsfA6a T-DNA knock-out line was identified. Thermoacquisition and HS-induction sHSP genes expression were not interrupted in the hsfA6a-KO line. Using the Motifsampler program to analyze three azetidine up-regulation sHSP-CI genes promoter, a non HSE with the conserved element, “GCGTTCA”, was identified. In promoter::GUS assay, confirmation of this element may play an important role in the HS response. In this study, we set up the expression profile of sHSPs and HSFs under HS and chemical inducers. But the individual HSF knock-out lines and other mutants are necessary to used to clarify the regulation of sHSPs expression by HSFs.