Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Organophosphorus compounds such as methyl-parathion are used in the various stages of production to control pests both in agricultural activity as in aquaculture. The mechanism of action of this type of compound is the inhibition of the enzyme acetylcholinesterase. The zebrafish (Danio rerio) has been increasingly used as an experimental model in varied fields such as development, genetics and pharmacological research. The methyl-parathion has been characterized as endocrine disruptor of the hypothalamic-pituitary-interrenal axis (HHI). An experiment was carried out of 96 hours of exposure of adult fish to the substance tested, at the concentration of 5.2 mg/L. Was evaluated the whole-body cortisol level in order to measure the endocrine response to a stressful event. Were also investigated the gene expression of glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR) and heat shock protein 70 (HSP 70). Fish exposed that have undergone a stressor event demonstrated low levels of cortisol. In addition, the fish stressed and exposed to agro-chemical showed a decreased expression of the StAR, HSP 70 and GR genes. The data indicate that exposure to methyl-parathion causes a decrease in the ability to respond appropriately to a stressor. Fish that have an inability to produce a satisfactory answer by the HHI axis are not able to make the necessary metabolic and ion adjustments for recovery the homeostasis, getting vulnerable to stress caused by aquaculture practices or environmental changes. / Compostos organofosforados como o parationato metílico são utilizados nas diversas etapas de produção para controlar pragas tanto na atividade agrícola como na aquicultura. O mecanismo de ação desse tipo de composto é a inibição da enzima acetilcolinesterase. O peixe-zebra (Danio rerio) vem sendo cada vez mais usado como modelo experimental em variados campos, como desenvolvimento, genética e pesquisa farmacológica. O parationato metílico já foi caracterizado como interruptor endócrino do eixo hipotálamo-hipófise-interrenal (HHI). Foi realizado um experimento de 96 horas de exposição de peixes adultos à substância testada, na concentração de 5,2 mg/L. Foi avaliado o nível de cortisol de corpo inteiro, visando medir a resposta endócrina a um evento estressor. Também foram investigadas a expressão dos genes do receptor de glicocorticoide (GR), da proteína regulatória de esteroidogenese aguda (StAR) e a proteína do choque térmico 70 (HSP 70). Os peixes expostos que foram submetidos a um evento estressor demonstraram baixos níveis de cortisol de corpo inteiro. Além disso, os peixes estressados e expostos ao agroquímico apresentaram uma diminuição da expressão dos genes GR, StAR e HSP 70. Os dados indicam que a exposição ao parationato metílico provoca uma diminuição da capacidade de responder adequadamente a um evento estressor. Peixes que possuem uma incapacidade em produzir uma resposta satisfatória do eixo HHI, não são capazes de realizar os ajustes iônicos e metabólicos necessários à recuperação da homeostase, ficando vulneráveis ao estresse causado pelas práticas aquícolas ou por alterações ambientais.

Made available in DSpace on 2016-12-08T15:59:06Z (GMT). No. of bitstreams: 1 Rafaella Santos.pdf: 19865129 bytes, checksum: eb985146ffff941f87977dbb7f5d7b13 (MD5) Previous issue date: 2015-07-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The 72kDa heat shock proteins are synthesized in our cells and are exported to the blood (eHSP72) in situations changed as oxidative stress and cardiovascular disease. However, both eHSP72 levels as impaired endothelial function has been associated with worse cardiovascular prognosis, including increased mortality. However, little is known about the association of these variables with 24h blood pressure in hypertensive. The aim of this study was to investigate the association between 24h blood pressure and levels of plasma eHSP72, oxidative stress parameters, endotelial function, body composition and physical activity in hypertensive patients. Methods: This is a cross-sectional study which evaluated 140 hypertensive patients (age 61 } 11.1 years) treated and followed by a Basic Health Unit. Were analyzed: 24h blood pressure, level of physical activity, usual by pedometer and endothelial function by plethysmography. In plasma concentrations were evaluated eHSP72 (by ELISA), the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) and lipid peroxidation levels (TBARS). Results: Hypertensive patients were classified as insufficiently active (8089.7 } 4287.9 steps/day). A binary logistic regression showed that those with overweight and obesity are more likely to have daytime systolic blood pressure (SBP) increased wakefulness compared to eutrophic. The same analysis also showed that the increase in vasodilatory capacity by reactive hyperemia (1ml/100ml tissue /min) reduces the chances of having daytime SBP change. Another important finding was that hypertensive patients with detectable eHSP72 were more likely to have nocturnal dipping present. There was no association between the variables of 24h blood pressure and the variables of oxidative stress. Conclusion: altered anthropometric indicators are associated with the daytime SBP uncontrolled, which may be indicative of cardiovascular overload. The night-time fall of blood pressure during sleep is associated with the expression of eHSP72, indicating that this protein may be associated in the modulation of a protective vascular function. / Proteínas de choque térmico de 72kDa são sintetizadas em nossas células e são exportadas para a corrente sanguínea (eHSP72) diante de situações como estresse oxidativo alterado e doenças cardiovasculares. Além disso, tanto os níveis de eHSP72 como a função endotelial prejudicada têm sido associados ao pior prognostico cardiovascular, incluindo aumento de mortalidade. No entanto, pouco se sabe sobre a associação destas variáveis com a pressão arterial de 24h em hipertensos. O objetivo deste estudo foi investigar a associação entre a pressão arterial de 24h e os níveis de eHSP72 plasmático, parâmetros de estresse oxidativo, função endotelial, composição corporal e o nível de atividade física em hipertensos. Métodos: Este é um estudo transversal no qual foram avaliados 140 hipertensos (idade 61±11,1 anos) tratados e acompanhados por uma Unidade básica de Saúde. Foram analisados: pressão arterial de 24h por monitorização ambulatorial (MAPA), nível de atividade física habitual por pedômetro e função endotelial por pletismografia. No plasma foram avaliadas as concentrações de eHSP72 (por ELISA), a atividade de enzimas antioxidantes catalase (CAT) e superóxido dismutase (SOD) e os níveis de lipoperoxidação (TBARS). Resultados: Os hipertensos foram classificados como pouco ativos (8089,7±4287,9 passos/dia). A regressão logística binária apresentou que aqueles com sobrepeso e obesidade têm mais chances de ter pressão arterial sistólica (PAS) da vigília aumentada em relação aos eutróficos. A mesma análise mostrou ainda que o aumento da capacidade vasodilatadora por hiperemia reativa (1 ml/100ml de tecido/min) reduz as chances de ter a PAS da vigília alterada. Outro achado de destaque e também inovador foi que hipertensos com eHSP72 detectável apresentaram mais chances de ter o descenso noturno sistólico presente. Não houve associação entre as variáveis da pressão arterial de 24h e as variáveis de estresse oxidativo. Conclusão: Indicadores antropométricos alterados se associam com a PAS da vigília descontrolada, o que pode ser um indicativo de sobrecarga cardiovascular. A queda da pressão arterial no período do sono está associada com a expressão de eHSP72, indicativo de que esta proteína pode estar envolvida como protetora na modulação da função vascular.

Mitochondria are essential eukaryotic organelles, acting as the sites for numerous crucial metabolic and signalling pathways. The biogenesis of mitochondria requires efficient targeting of several hundreds of proteins from the cytosol, to their varied functional locations within the organelle. The translocation of localized proteins across the inner membrane, and their subsequent folding is achieved by the ATP-dependent function of mitochondrial Hsp70 (mtHsp70). It is a bonafide member of the Hsp70 chaperone family, which are involved in a multitude of functions, together aimed at protein quality control and maintenance of cellular homeostasis. These varied functions of Hsp70 proteins require binding to exposed hydrophobic patches in substrate polypeptides thus preventing non-productive associations. The interaction with substrates occurs through the substrate-binding domain (SBD) and is regulated by the ATPase activity of the nucleotide-binding domain (NBD), through a series of conformational changes. Conversely, substrate binding to the SBD also stimulates ATP hydrolysis, and thereby the core activities of the two domains are regulated by mutual allosteric signalling. This mechanism of bidirectional inter-domain communication is indispensable for Hsp70 function, which is characterized by cycles of substrate binding and release, coupled to cycles of ATP binding and hydrolysis. The process of allosteric regulation in Hsp70 proteins has been comprehensively investigated, especially in the bacterial homolog, DnaK. However, the in vivo functional significance of inter-domain communication in the eukaryotic mtHsp70 system and the mechanism of its regulation remain unexplored. Furthermore, the complex physiological implications of impairment in allosteric communication and their correlation with diverse disease conditions, including Myelodysplastic syndrome (MDS), and Parkinson’s disease (PD), are yet to be elucidated. Based on this brief introduction, the primary research objectives set out in the present thesis were to: 1. uncover the regulation of ligand-modulated allosteric communication between the two domains of mtHsp70; and its in vivo significance in the context of protein import into the organelle. (Chapter 2) 2. understand the role of mtHsp70 in progression of Parkinson’s disease; and to study the modulation of α-synuclein toxicity by the protein quality control function of the mtHsp70 chaperone network. (Chapters 3 and 4) We have employed a battery of genetic and biochemical approaches to investigate the above questions using the Saccharomyces cerevisiae mtHsp70 protein, Ssc1; an essential protein that is involved in a plethora of critical functions in this eukaryotic model system. Objective 1: Structural studies, primarily in bacterial DnaK, have yielded mechanistic insights into its interactions with ligands and cochaperones, as well as conformational transitions in different ligand-bound states. In recent years, the availability of crystal structures of full-length DnaK and detailed information from NMR studies and single-molecule resolution spectroscopic analyses (both DnaK and eukaryotic Hsp70s), have significantly contributed to our understanding of the inter-domain interface, critical residues and contacts, and the energetics of the entire process of ligand-modulated conformational changes. Although eukaryotic mtHsp70s have a high degree of conservation with DnaK, they possess significant differences in their conformational and biochemical properties. They are essential for a vast repertoire of physiological functions, which are distinctly different from their bacterial counterpart. Using a combined in vivo and in vitro approach, we have uncovered specific structural elements within mtHsp70s, which are required for allosteric modulation of the chaperone cycle and maintenance of in vivo functions of the protein. Foremost, we demonstrate that a conserved SBD loop, L4,5 plays a critical role in inter-domain communication, and multiple mutations in this loop result in significant growth and protein translocation defects. The mutants are associated with a specific set of altered biochemical properties, which are indicative of impaired inter-domain communication. Using the loop L4,5 mutant, E467A as a template for genetic screening, we report a series of intragenic suppressor mutations, which are capable of correcting a distinct subset of the altered properties, and thereby leading to restoration of in vivo functions, including growth, preprotein import and mitochondria biogenesis. The suppressors modify the altered conformational landscape associated with E467A, and also provide us with information regarding unique aspects governing the regulation of allosteric communication, especially in physiological contexts. Strikingly, they reveal that restoration of communication in the NBD to SBD direction is sufficient for function, when the protein is primed in a high ATPase activity state. In this unique scenario, the requirement for ATPase stimulation upon substrate binding is rendered unnecessary, thereby making conformational changes in the SBD to NBD direction, dispensable for function. Further, we provide evidence to show that loop L4,5 functions synergistically with the linker region, working in tandem for organization of the inter-domain interface and propagation of communication. Together, our analyses provide the first insights into regulation of allosteric inter-domain communication in vivo and their implications in mitochondrial protein translocation and organelle biogenesis. Objective 2: Point mutations in the loop L4,5 have been associated with Myelodysplastic syndrome. Additionally, a mutation isolated in clinical cases of Parkinson’s disease was found to be impaired in allosteric communication. These observations further highlight the importance of efficient inter-domain communication in mtHsp70 in the complex physiological scenario of eukaryotic cells. Independent clinical screens of PD patients have revealed unique point mutations in the mtHsp70 and a strong association of the gene locus with the disease progression. This is also correlated with decreased mtHsp70 levels in affected neurons and the interactions of this protein with established PD-candidate proteins like α-synuclein and Dj-1. Further, mitochondrial dysfunction is a common phenomenon associated with neurodegenerative disorders. To understand the specific role of mtHsp70 in PD, we have developed a yeast model for studying the disease variants in isolation from other players of the multifactorial disease, and in complete absence of the wild type protein. We generated two analogous PD-mutations in Ssc1, R103W and P486S; which recapitulated the symptoms of mitochondrial dysfunction in affected neurons, including cell death, inner membrane depolarization, increased generation of ROS, and respiratory incompetence. At the molecular level, we observed an increased aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein cochaperone partners thereby resulting in loss of chaperoning activity and impaired mitochondrial protein quality control. Remarkably, these altered biochemical properties mimicked similar defects in the human mtHsp70 variants, therefore, affirming the involvement of mtHsp70 in PD progression. To further investigate the relevance of impaired mitochondrial protein quality control in PD, we have explored whether mtHsp70 can act as a genetic modifier of α-synuclein toxicity. It is known that α-synuclein can act as an unfolded substrate for the Hsp70 chaperone system and also deposits as intracellular aggregates in PD-affected brains. Intriguingly, it is known to translocate into mitochondria under conditions of neuronal stress in spite of lacking a canonical mitochondrial signal sequence. Utilizing our yeast-PD model, we find that targeting of α-synuclein A30P disease variant into mitochondria leads to a severe mitochondrial dysfunction phenotype in the wild type Ssc1 background, but not the P486S mutant background. This results in multiple cellular manifestations, which are reversed upon overexpression of the Ssc1 chaperone. Significantly, increasing the J-protein cochaperone availability also leads to reversal of the mutant-associated defects. However, the simultaneous overexpression of both together does not additively improve the protective effects; highlighting the importance of the relative availability of chaperone and cochaperone proteins in preventing aggregation. Our analyses further reveal that while both the wild type and P486S Ssc1 proteins are equally capable of delaying aggregation of α-synuclein, only the wild-type chaperone is better able to prevent aggregation in the presence of its J-protein cochaperone, leading to accumulation of soluble oligomeric species. These observations raised the intriguing possibility, that the reduced chaperoning ability of the proline to serine PD-mutant is, in fact, a compensatory adaptation, favoring the aggregation of α-synuclein over its more toxic soluble oligomeric form. We verify this hypothesis with the aggregation kinetics of A30P α-synuclein, whose intrinsically lower aggregation tendency results in a pronounced delay in aggregation with the wild-type chaperone, thereby strongly favoring the toxic oligomeric species and correlating with the observed lethality in yeast cells. In conclusion, our study provides a model of α-synuclein aggregation-related toxicity and its modulation by the extent of protein quality control within the mitochondrial matrix, through the action of the mtHsp70 chaperone network.

Rotavirus is one of the leading etiological agents of gastroenteritis in young of many species including humans worldwide and is responsible for about 600,000 infant deaths per annum. Rotavirus belongs to the Reoviridae family, and its genome is composed of 11 double-stranded RNA segments that encode six structural proteins and six nonstructural proteins. Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm¬like structures. A recent study suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP5, the translation product of gene segment 11 has a predicted molecular eight of 21 kDa. NSP5 has been reported to exist in multiple isoforms ranging in size from 28-and 32-35 kDa from a 26-kDa precursor has been attributed to O-glycosylation and hyperphosphorylation. To study different properties of the protein, recombinant NSP5 containing an N-terminal hisidine tag was expressed in bacteria and purified by affinity chromatography. A significant observation was the similarity in phosphorylation property of the bacterially expressed and that expressed in mammalian cells. While the untagged recombinant protein failed to undergo phosphorylation in vitro, addition of His tag or deletions at the N-terminus promoted phosphorylation of the protein in vitro, which is very similar to the reported properties exhibited by the corresponding proteins expressed in mammalian cells. Phosphorylation of NSP5 in vitro is independent of the cell type from which the extract is derived suggesting that the kinases that phosphorylate NSP5 are distributed in all cell types. Among the C-terminal deletion mutants studied, NH-∆C5 and NH-∆C10 were phosphorylated better than full-length NSP5, but NH-∆C25 and NH¬∆C35 showed substantial reduction in the level of phosphorylation compared to full-length NSP5. These results indicate that the C-terminal 30 residues spanning the predicted α-helical domain of NSP5 are critical for its phosphorylation in vitro which is in correspondence with previous findings that C-terminal 21 amino acids of NSP5 direct its insolubility, hyperphosphorylation, and VLS formation. The results revealed that though the tagged full-length and some of the mutants could be phosphorylated in vitro, they are not suitable substrates for hyperphosphorylation unlike the similar proteins expressed in mammalian cells or infected cells. Analysis by western blot and mass spectrometry revealed that the bacterially expressed NH-NSP5 is indeed phosphorylated. It appears that prior phosphorylation in bacteria renders the protein conformationally not amendable for hyperphosphorylation by cellular kinases in vitro. Mutation of the highly conserved proline marginally enhanced its phosphorylation in vitro but the stability of protein is affected. Notably, mutation of S67A, identified as a critical residue for the putative caesin kinase-I and-II pathways of NSP5 phosphorylation, affected neither the phosphorylation nor the ATPase activity of NSP5. These results suggest that bacterially expressed NSP5 by itself has undectable auto-kinase activity and it is hypophosphorylated. Purified recombinant NSP5 has been reported to possess an Mg¬ 2+-dependent ATP-specific triphosphatase activity. The results indicated that deletion of either C-terminal 48 amino acids or N-terminal 33 residues severely affected the ATPase activity of recombinant NSP5, underlying the importance of both N-and C-terminal domains for NSP5 ATP hydrolysis function. NSP5 expressed in rotavirus infected cells exists as inter-molecular disulfide-linked dimeric forms and it appears that the 46 kDa isoforms, that are phosphorylated, corresponds to dimer as revealed by western blotting. Analytical gel filtration analysis of NH-NSP5, NH-ΔN43 and NH-ΔN33-ΔC25 showed most of the proteins in void volume, but an additional peak corresponding to the mass of dimeric species further supports that NSP5 is basically a dimer that undergoes oligomerization. Analysis by sucrose-gradient fractionation revealed that NH-NSP5 and NH-ΔN43 proteins were mainly distributed in the lower fraction of the gradient suggesting the existence of high molecular weight complexes or higher oligomers. The multimeric nature of NSP5 and its mutants was further confirmed by dynamic light scattering which suggests that high molecular weight complexes are of homogenous species. The correlation curves showed a low polydispersity distribution and a globular nature of recombinant NH-NSP5 proteins. The present results clearly demonstrate that dimer is the basic structural unit of NSP5 which undergoes oligomerization to form a complex consisting of about 20-21 dimers. The nonstructural protein 5 is hyperphosphorylated in infected cells and cellular kinases have been implicated to be involved in its phosphorylation. NSP5 contains multiple consensus sites for phosphorylation by several kinases, but the cellular kinases that specifically phosphorylate NSP5 in infected cells are yet to be identified. Previous studies from our laboratory using signaling pathway inhibitors revealed that recombinant NH¬NSP5 and its deletion mutants can be phosphorylated in vitro by purified cellular kinases and by mammalian cell extracts. These studies also showed the involvement of PI3K-Akt and MAPK signaling pathways in NSP5 phosphorylation and a negative role for GSK3β in the phosphorylation of bacterially expressed recombinant NSP5 in vitro. In the present work, using phospho-specific anti-Ser9 GSK3β antibody, we observed that GSK3β is inactivated in a rotavirus infected MA104 cells in a strain-independent manner. GSK3β¬specific small interfering RNA (siRNA-GSK3β) reduced GSK3β levels leading to increased level of synthesis of the structural rotavirus protein VP6 and NSP5 hyperphosphorylation compared to control siRNA. The pharmacological kinase inhibitors (LY294002, Genistein, PD98059, and Rapamycin) studies at the concentrations tested did not significantly affect rotavirus infection as seen from the number foci, while U0126 severely affected rotavirus replication. The results clearly demonstrated the importance of the MEK1/2 signaling pathway in the successful replication of rotavirus and NSP5 hyperphosphorylation in rotavirus-infected cells. In contrast inhibition of GSK3β activity by LiCl, increased in general, the number of foci by greater than 2-fold for all viral strains studied. These results suggest that MEK1/2 pathway majorly contributes to GSK3β inactivation in rotavirus infected cells. Thus, our results reveal that rotavirus activates both the PI3K/Akt and FAK/ERK1/2 MAPK pathways and appears to utilize them as a strategy to activate mTOR, and inhibit GSK3β through phosphorylation on serine 9, the negative regulator of rotavirus NSP5 phosphorylation, and thus facilitate translational competence of rotaviral mRNAs during virus replication cycle. It was shown previously in the laboratory by co-immunoprecipitation assay that Hsp70 interacts with rotaviral proteins VP1 and VP4 in rotavirus-infected mammalian cells. In this study, the interactions between Hsp70 with VP1 and VP4 were further evaluated in vitro by GST-pull down assay. It was observed that the N-terminal ATPase and C-terminal peptide-binding domain of Hsp70 is necessary for its direct interaction with VP1 and VP4. The presence of Hsp70 in purified double-and triple-layered virus particles further supported the observed interactions of rotaviral proteins VP1 and VP4 with Hsp70. However, the specific interaction observed between Hsp70 and rotaviral capsid proteins, VP1 and VP4 in viral particles suggests that Hsp70 has an important role during rotavirus assembly which requires further investigation.