As Hsp70 são proteínas centrais no sistema de homeostasia proteica da célula. Pelo fato delas estarem envolvidas em uma grande variedade de processos relacionados ao enovelamento correto das proteínas, elas estão envolvidas em processos como envelhecimento, doenças degenerativas, como Alzheimer, e alguns tipos de câncer. Uma das etapas essenciais no seu ciclo funcional é a troca de ADP por ATP, um processo que é acelerado pelos fatores de troca de nucleotídeos (NEFs) que, em bactérias e mitocôndrias, correspondem à proteína GrpE. Por razões ainda não bem compreendidas, duas isoformas estão presentes nas mitocôndrias de humanos, a GRPEL1 e a GRPEL2. Pouco se sabe da dinâmica da interação destas com a Hsp70 mitocondrial de humanos (mortalina) porque havia uma dificuldade em se obter esta proteína na sua forma solúvel e funcional (atualmente superada). Dessa forma, o presente trabalho de pesquisa busca caracterizar os aspectos bioquímicos e biofísicos dessas proteínas junto à mortalina, visando compreender a dinâmica da interação entre elas, contribuir para a elucidação da rede de interações das Hsp70 e compreender o porquê de 2 isoformas estarem presentes em mamíferos. Para isto, ensaios in vitro das proteínas mortalina, GRPEL1 e GRPEL2 recombinantes foram realizados. Elas foram expressas e purificadas por cromatografia de afinidade ao Ni2+ e gel filtração. As GrpEs tiveram seus graus de pureza e enovelamento correto avaliadas por SDS-PAGE e dicroísmo circular. Suas estruturas terciárias e quaternárias foram avaliadas através da cromatografia de exclusão molecular analítica e do crosslinking químico. Com as proteínas tendo sido purificadas, ensaios de interação molecular foram realizados através do pulldown, do ITC e, adicionalmente, foram feitos ensaios de agregação para investigar um possível papel das GrpEs no processo de agregação térmica da mortalina. Todas as proteínas puderam ser obtidas solúveis e com alto grau de pureza. Os ensaios de pulldown validaram a interação entre a mortalina e as GrpEs, mas essas interações não foram detectadas no ITC. Por fim, não houveram evidências de que as GrpEs atuem no sentido de prevenir a agregação térmica da mortalina. / Hsp70 are proteins that play a central role in cellular protein homeostasis. Because they are involved in a variety of processes related to protein folding, they are also involved in processes such as aging, degenerative diseases like Alzheimer and certain types of cancer. One of the essential steps in the Hsp70 functional cycle is the exchange of ADP for ATP, a process accelerated by the nucleotide exchange factors (NEF´s) which, in bacteria and mitochondria, corresponds to GrpE protein. For reasons not well understood yet, two isoforms are present on human mitochondria, GRPEL1 and GRPEL2. Little is known about the dynamics of their interaction with human mitochondrial Hsp70 (mortalin) because it was difficult to produce this protein in its soluble and functional form (now overcome by co-expression strategies with one co-chaperone). That being said, the current research work seeks to characterize the biochemical and biophysical aspects of those proteins together with mortalin in order to comprehend the dynamics of their interaction, to contribute on the elucidation of the Hsp70 interaction network and to comprehend why two isoforms are present. For this, in vitro assays of the recombinant proteins mortalin, GRPEL1 and GRPEL2 were carried out. They were expressed and purified by Ni2+ affinity chromatography and gel filtration. Both GrpEs had their degree of purity and correct folding assessed by SDS-PAGE and circular dichroism. Their tertiary and quaternary structures were evaluated by analytical size exclusion chromatography and chemical crosslinking. Having the proteins being purified, molecular interactions assays were done with pulldown, ITC and, additionally, aggregation assays were carried out to investigate a possible role played by GrpEs in the thermal aggregation process of mortalin. All the proteins could be obtained soluble and with a high degree of purity. Pull-down assays validated the interaction between mortalin and GrpEs, but this interaction could not be detected by ITC. Lastly, there was no evidence that GrpEs acted out preventing the thermal aggregation process of mortalin.

As chaperonas moleculares da família Hsp70 desempenham funções cruciais nas células de todos os organismos vivos, de procariotos a eucariotos. Nestes, estão presentes em todos os compartimentos celulares e nas mitocôndrias é expressa uma isoforma própria (mtHsp70), que participa dos processos enovelamento e maturação de proteínas bem como de sua importação para a matriz mitocondrial. Diante da crescente demanda de pesquisa sobre doenças tropicais negligenciadas, foi tomado como objeto de estudo neste trabalho uma Hsp70 mitocondrial de Leishmania braziliensis (LbmtHsp70) com o intuito de caracterizá-la estrutural e funcionalmente em comparação à ortóloga humana com maior identidade: a mtHsp70 também chamada de mortalina, GRP75, HspA9 ou PBP74. A LbmtHsp70 foi purificada em sua forma enovelada, em sistema monodisperso apresentando dados hidrodinâmicos condizentes com a forma monomérica, foi testada sua estabilidade quanto à influência de nucleotídeos de adenosina (ATP e ADP) à sua estrutura e, por fim, foram feitos ensaios para avaliar sua atividade ATPásica e de energia de interação com nucleotídeos. De forma geral, a LbmtHsp70 é bastante similar à mortalina, como pode ser evidenciado pelos resultados obtidos com algumas particularidades. / The molecular chaperones from the Hsp70 family perform critical cell roles in all organisms, from prokaryotes to eukaryotes. In the last ones, they are found in all cell compartments and a particular isoform is expressed in the mitochondria, where it carries out folding and maturation processes as well as the import of proteins to the mitochondrial matrix. In face of the growing demand for research about neglected tropical diseases, in this study a Hsp70 from Leishmania braziliensis\'s mitochondria was taken as object of study for further structural and functional characterization in comparison to the human orthologous which presents the highest identity to LbmtHsp70: the mtHsp70 also known as mortalin, GRP75, HspA9 or PB74. LbmtHsp70 was obtained in folded state in monodisperse system with hydrodynamic data consistent to monomeric conformer, stability and adenosine nucleotides influence to its structure were analyzed, and were performed assays for ATPase activity and nucleotide interaction energy. In a general way LbmtHsp70 is very similar to mortalin as can be shown through the results, but with some peculiarities.

As chaperonas moleculares da família Hsp70 desempenham funções cruciais nas células de todos os organismos vivos, de procariotos a eucariotos. Nestes, estão presentes em todos os compartimentos celulares e nas mitocôndrias é expressa uma isoforma própria (mtHsp70), que participa dos processos enovelamento e maturação de proteínas bem como de sua importação para a matriz mitocondrial. Diante da crescente demanda de pesquisa sobre doenças tropicais negligenciadas, foi tomado como objeto de estudo neste trabalho uma Hsp70 mitocondrial de Leishmania braziliensis (LbmtHsp70) com o intuito de caracterizá-la estrutural e funcionalmente em comparação à ortóloga humana com maior identidade: a mtHsp70 também chamada de mortalina, GRP75, HspA9 ou PBP74. A LbmtHsp70 foi purificada em sua forma enovelada, em sistema monodisperso apresentando dados hidrodinâmicos condizentes com a forma monomérica, foi testada sua estabilidade quanto à influência de nucleotídeos de adenosina (ATP e ADP) à sua estrutura e, por fim, foram feitos ensaios para avaliar sua atividade ATPásica e de energia de interação com nucleotídeos. De forma geral, a LbmtHsp70 é bastante similar à mortalina, como pode ser evidenciado pelos resultados obtidos com algumas particularidades. / The molecular chaperones from the Hsp70 family perform critical cell roles in all organisms, from prokaryotes to eukaryotes. In the last ones, they are found in all cell compartments and a particular isoform is expressed in the mitochondria, where it carries out folding and maturation processes as well as the import of proteins to the mitochondrial matrix. In face of the growing demand for research about neglected tropical diseases, in this study a Hsp70 from Leishmania braziliensis\'s mitochondria was taken as object of study for further structural and functional characterization in comparison to the human orthologous which presents the highest identity to LbmtHsp70: the mtHsp70 also known as mortalin, GRP75, HspA9 or PB74. LbmtHsp70 was obtained in folded state in monodisperse system with hydrodynamic data consistent to monomeric conformer, stability and adenosine nucleotides influence to its structure were analyzed, and were performed assays for ATPase activity and nucleotide interaction energy. In a general way LbmtHsp70 is very similar to mortalin as can be shown through the results, but with some peculiarities.

This thesis is composed of two topics, for which trypanosomatids and evolution are common denominators. First part deals with phylogenetic relationships among monoxenous trypanosomatids, with emphasis on flagellates parasitizing dipteran hosts, analyzed mainly from biogeographical and evolutionary perspectives. Second part focuses on the trypanosomatid Trypanosoma brucei, causative agent of severe diseases, which serves as a model organism for functional studies of evolutionary conserved mitochondrial proteins, in particular those involved in replication, maintenance and expression of the mitochondrial genome, also termed the kinetoplast. This thesis identified the mtHsp70/mtHsp40 chaperone machinery as an essential component of replication and maintenance of the kinetoplast, and also identified numerous conditions under which mtHsp70 has a tendency to aggregate. Moreover, several conserved proteins, previously identified to be part of the mitochondrial ribosome, were shown to be important for translation of the mitochondrial transcripts.

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.

The role of mitochondria is multidimensional and ranges in vast areas, including apoptosis, cellular response towards stress, metabolism, which is regulated by a plethora of proteins, acting together to maintain cellular and organellar homeostasis. In spite of the presence of mitochondrial DNA, most of the mitochondrial proteins are nuclear encoded and translocated inside the organelle through dedicated translocases present on outer and inner membrane of mitochondria. To fulfil the cellular energy demand, mitochondria efficiently generate ATP by oxidative phosphorylation, and thus are considered as "power house of cell." There occurs a transfer of electrons from various oxidizable substrates to oxygen, which is achieved by a series of redox reactions with generation of water as a byproduct. This process is coupled with ATP synthesis, involves five protein-complexes present in the inner mitochondrial membrane. During this process, it generates extremely reactive intermediate species of oxygen as a byproduct collectively referred as Reactive Oxygen Species (ROS) through partial reduction of oxygen. These intermediate metabolites of oxygen include superoxide anion (O2-º), H2O2 and highly reactive hydroxyl radicals (OHº). Although ROS are produced by different cellular sources, such as widely expressed and evolutionary conserved NADPH Oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases and cytochrome P450 enzymes but mitochondria are one of the major contributors of cellular ROS. Earlier, reactive oxygen species were considered as harmful but for past few decades, the role ROS has been appreciated as signalling molecules. Because of their high reactivity, these species can cause redox mediated modifications to cellular components and thus have an ability to participate in signalling process. The regulation of signalling pathway by ROS is governed by either alterations in cellular redox conditions or by oxidative modifications of certain residues in proteins, which are involved in signalling cascades. Reactive Oxygen Species can modify amino acid residues, interact with Fe-S clusters or other metal complexes and induce dimerization of proteins to alter protein structure and function. ROS causes modifications to critical amino acids, mainly by oxidation of cysteine residues, where oxidation of sulfhydryl group (-SH) of a single cysteine residue leads to formation of sulfenic (-SOH), sulfinic (-SO2H), sulfonic (-SO3H), or S-glutathionylated (-SSG) derivatives. Thus, by incorporating these modifications, ROS affects the function of proteins, thereby modulating the cellular signalling process. On the other hand, the accumulation of higher level of reactive oxygen species may damage cellular components causing oxidative stress. Therefore, it is necessary to maintain the ROS levels and regulation of intracellular redox homeostasis depends upon a complex network of antioxidant molecules. These antioxidants range from low molecular weight glutathione to large proteins like glutathione peroxidases. Cell has an array of antioxidants with different subcellular locations. Superoxide Dismutase which catalyzes dismutation of superoxides and converts them to H2O2, localizes in cytosol, mitochondrial intermembrane space and extracellular matrix. Different isoforms of Glutatione Peroxidases (GPx) and Peroxiredoxins (Prx) are located in cytosol as well as in mitochondria and scavenge H2O2 by using glutathione (GSH) and thioredoxin (Trx) respectively, as co-factors. During this peroxidase activity of GPx and Prx, GSH and Trx get oxidized and recycled back to the reduced form by Glutathione Reductase (GR) and Thioredoxin Reductase (TR) correspondingly, with the help of NADPH. Thus, GPx system (GPx, GR, GSH and NADPH) and Prx system (Prx, Trx, TR and NADPH) helps in maintenance of redox balance by scavenging H2O2. Catalase is present in peroxisomes for the catalytic degradation of H2O2. Along with Thioredoxin, glutaredoxin (Grx) also reduces protein disulphides and maintains the redox homeostasis. Although, reactive oxygen species are important for normal physiological process, oxidative stress caused by imbalanced ROS levels is thought to be involved in progression of many disorders. However, in most of the diseases, the role of ROS is not yet clear. Elevated oxidative stress is observed with insulin resistance and progression of type II diabetes mellitus, and the resultant high glucose levels alter mitochondrial physiology, leading to the fragmentation of organelle. However, on contrary it has also been observed that ROS improves insulin sensitivity. ROS is directly involved in progression of neurodegenerative disorders, which are characterized by oxidative stress mediated neuronal loss. Interestingly, in case of cancer ROS plays a differential role. At moderately higher levels, ROS helps cancer cells to detach from the matrix and thus assist in metastasis but the higher accumulation of ROS leads to oxidative stress mediated cell death. Thus, cancer cells have an enhanced expression level of antioxidants to maintain the optimum ROS concentration for their survival and proliferation. The role of ROS in cellular signalling and progression of diseases highlights the importance of redox regulation. Mitochondria being the major source of ROS, harbours various redox regulators such as a mitochondrial permeability transition pore (mPTP), inner membrane anion channel (IMAC), Ca++ ions, etc. In addition, certain proteins like Hsp31/DJ1 class also translocate into the organelle in a stress dependent manner to maintain redox homeostasis. These proteins are encoded by the nuclear genome and translocated in the organelle, suggesting the importance of mitochondrial import machinery in regulation of redox balance. Another such example is MIA pathway of protein import, where MIA40 regulates ROS indirectly by catalyzing folding of disulfide containing proteins such as SOD-1 in a redox coupled process. However, under most cases, the physiological disorders lead to uncontrolled production of reactive oxygen species, thereby overloading the cellular antioxidant defence machinery. The failure of the antioxidant machinery leads to enhanced disease progression. Under such disease conditions where the upheaval of redox homeostasis leads to the accumulation of ROS, artificial antioxidants can be used to protect cells against oxidative damage. Artificial systems such as Cyclodextrins, metal complexes, porphyrins, polymers, supramolecules and biomolecules such as nucleic acids, catalytic antibodies and proteins, have been created to mimic the structures and functions of natural enzymes through various approaches. In the present thesis, we have elucidated the role of two mitochondrial proteins, which are part of mitochondrial import motor, as redox regulators and the effect of artificial antioxidants in maintenance of redox homeostasis under stress. A detailed description on importance of ROS in cellular signalling and disease progression has been included in Chapter I, which gives a preface for the work mentioned in this thesis. Chapter II to chapter V elucidates the main objectives of the present thesis, which are: 1. Identification of novel human mitochondrial regulators of redox homeostasis • Role of NEF in redox sensing (Chapter II) • Evolved function of J-like protein in ROS regulation (Chapter III) 2. Characterization of potential artificial antioxidants as redox therapeutics • Organo-selenium compounds as potential artificial antioxidants (Chapter IV) • Use of nanoparticles as a natural antioxidant mimics (Chapter V) Chapter II: Mitochondrial Hsp70 (mtHsp70) plays a critical role for the import of the precursor proteins. The import activity of mtHsp70 is attributed by cyclic binding and release of precursor proteins which in turn is regulated by co-chaperones J-proteins and nucleotide exchange factor (NEF). The affinity for substrate is governed by the binding of ADP or ATP at the N-terminal nucleotide binding pocket of mtHsp70. The affinity for substrate is higher in ADP bound state as compared to ATP bound state. mtHsp70 by its ATPase activity hydrolyze ATP (low-affinity state) to ADP (high-affinity state), which is replaced back to ATP by NEF thus maintaining the mtHsp70 cycle for protein import. In the present study, we have biochemically and functionally characterized GrpEL1 and GrpEL2 as a nucleotide exchange factor for mtHsp70. We observed that like their yeast ortholog Mge1, both the mammalian NEFs interacts with mtHsp70 and exchange ADP from ATP to maintain the cycle of mtHsp70. Interestingly, we observed that both the NEFs are part of human mitochondrial import motor and are recruited at the import motor as hetero-subcomplex. The formation of GrpEL1-EL2 hetero-subcomplex is important to maintain the stability of both the NEFs. In this study, we have elucidated that the interplay between the two NEFs governs organellar response towards oxidative stress. Chapter III: Redox imbalance generates multiple cellular damages leading to oxidative stress mediated pathological conditions such as neurodegenerative diseases, diabetes, ageing and cancer progression. Therefore, maintenance of ROS homeostasis is most important, that involves well-defined antioxidant machinery. In the present chapter, we have identified for first time a component of mammalian protein translocation machinery, Magmas, to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues, cancer cells and tissues of developmental origin that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance towards oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of ETC-complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as an ROS sensor was found to be independent of its role in protein import, underlying its dual role in human mitochondria. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cellular tolerance to oxidative stress even in yeast model organism. The cyto-protective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress related diseases. Chapter IV: The dysregulation of antioxidant machinery in oxidative stress mediated disorders lead to accumulation of excess ROS, highlighting the importance of artificial antioxidants. For the therapeutics of oxidative stress related disorders, artificial antioxidants have been used as combination redox therapy. In order to realize potent biocompatible antioxidants with minimum toxicity, we have utilized two approaches – synthesis of organic compounds and nanoparticle based enzyme mimetics. We have synthesized novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities, which provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being widely clinically evaluated by several research groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this study has the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.. Chapter V: Nanomaterials with enzyme-like properties have attracted significant interest, although limited information is available on their biological activities in cells. Here, we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme, glutathione peroxidase by using cellular glutathione as a co-factor. Although a bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origins (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species, when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.

My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.