Most proteins must fold into native three-dimensional structures to be functional. But, newly synthesized proteins are at high risk of misfolding and aggregating in the cell. Stress, disease or mutations can also cause protein aggregation. A cyanobacterial small heat shock protein, Hsp16.6, can act as a chaperone to prevent irreversible protein aggregation during heat stress. This thesis is focused on the properties of proteins that were associated with Hsp16.6 during heat stress, and which therefore may be “substrates” of Hsp16.6. Bioinformatics were used to determine if Hsp16.6 preferentially binds to proteins with certain properties, and biochemical studies were performed to investigate how the substrates actually behave with Hsp16.6 during heat stress. It was found that Hsp16.6 preferentially binds to proteins with higher molecular weight, higher acidity, higher percentage of charged residues (especially negatively charged residues), and a lower percentage of hydrophobic residues compared to all proteins encoded by the Synechocystis genome. Proteins bound to Hsp16.6 were also slightly enriched in VQL motifs. The potential substrate fructose bisphosphate aldolase class II (FBA) was expressed in E.coli and purified. FBA could be protected by Hsp16.6 from aggregation through forming a complex with Hsp16.6 during heat stress in vitro, consistent with it being a substrate of Hsp16.6. Another potential substrate, elongation factor G1 (EF-G1) was also expressed in E.coli and purified. EF-G1 did not form insoluble aggregates even at 47°C, but circular dichroism spectroscopy revealed the secondary structure has melted at this temperature, and the protein eluted earlier than unheated protein on size exclusion chromatography. Thus, EF-G1 appears heat sensitive, and may also be an in vivo substrate of Hsp16.6. Lastly, in vivo study studies were performed to determine the amount of FBA and EF-G1 in Synechocystis cells. Both proteins are abundant, with FBA levels (around 2% of total cell protein) being about twice that of EF-G1. Further in vivo experiments will be needed to confirm that FBA and EF-G1 are substrates of Hsp16.6.

Background: Oxidative stress and myocyte apoptosis are thought to play an important role in the pathogenesis, progression and prognosis of heart failure (HF). Heat shock protein 27 (Hsp27) has been found to confer resistance to oxidative stress in cultured cells; however, the role of Hsp27 in in-vivo hearts remains to be determined. Aim: To investigate the effects of Hsp27 over-expression on doxorubicin-induced HF. Methods and Results: Transgenic mice (TG) with cardiac specific over-expression of Hsp27 and their wild type littermates (WT) were challenged with doxorubicin (25 mg/kg, IP) to induce HF. At day 5, TG mice had significantly improved cardiac function and viability and decreased loss of heart weight following doxorubicin exposure compared with WT. In another parallel experiment, doxorubicin-induced increased levels of reactive oxygen species, protein carbonylation, apoptosis and morphologic changes were detected in the mitochondria in WT hearts, whereas these effects were markedly attenuated in TG hearts. In addition, upregulation of heat shock protein 70 and heme oxygenase-1 was present in the TG hearts after doxorubicin stimulation in comparison to WT hearts. Conclusion: These findings indicate that Hsp27 may play a key role in resistance to doxorubicin-induced cardiac dysfunction.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18095号 / 理博第3973号 / 新制||理||1573(附属図書館) / 30953 / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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

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.