Indiana University-Purdue University Indianapolis (IUPUI)

Abstract THE CHARACTERIZATION OF CHIMERIC CHAPERONE FLAGRP170 AS A NOVEL RADIOPROTECTANT By Tyler Nguyen, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2017 Major Director: Dr. Xiang-Yang (Shawn) Wang, Ph.D., Professor, Department of Human and Molecular Genetics Radiation therapy (RT) is restricted by toxic effects on adjacent normal tissue, which limits RT efficacy in cancer treatment. Damage to normal tissue, such as radiosensitive intestine and bone marrow compartments, results in acute radiation damage. To reduce normal tissue injury in the setting of RT, we examine the potential radioprotectant, Flagrp170, a chimeric protein. Flagrp170 is comprised of glucose-regulated protein-170 (Grp170) and a NF-κB activating sequence derived from flagellin. We show that Flagrp170 can protect normal tissues post irradiation, indicated by TUNEL and clonogenic assays. However, treatment with Flagrp170 does not influence tumor response to RT. Studies indicate that Flagrp170 activates the transcription factor NF-κB, a strong pro-survival signal. In addition, Flagrp170 can induce production of radioprotective cytokines as well. Data suggests that Flagrp170 has potential as a novel radioprotectant in the setting of RT. The combination of Flagrp170 therapy and RT may lead to improved treatment outcomes.

The type VI secretion system (T6SS) is a protein export pathway that mediates competition between Gram-negative bacteria by facilitating the injection of toxic effector proteins from attacking cells into target cells. To function properly, many T6SSs require at least one protein that possesses a proline-alanine-alanine-arginine (PAAR) domain. These PAAR domains are often found within large, multi-domain effectors that possess additional N- and C-terminal extension domains whose function in type VI secretion is not well understood. The work described herein uncovers the function of these accessory domains across multiple PAAR-containing effectors. First, I demonstrated that thousands of PAAR effectors possess N-terminal transmembrane domains (TMDs) and that these effectors require a family of molecular chaperones for stability in the cell prior to their export by the T6SS. Our findings are corroborated by co-crystal structures of chaperones in complex with the TMDs of their cognate effectors, capturing the first high-resolution structural snapshots of T6SS chaperone-effector interactions. Second, I characterize a previously undescribed prePAAR effector named Tas1. My work shows that the C-terminus of Tas1 possesses a toxin domain that pyrophosphorylates ADP and ATP to synthesize the nucleotides adenosine penta- and tetraphosphate (hereafter referred to as (p)ppApp). Delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ADP and ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. These findings reveal a new mechanism of interbacterial antagonism, the first characterization of a (p)ppApp synthetase and the first demonstration of a role for (p)ppApp in bacterial physiology. TMD- and toxin-containing PAAR proteins constitute a large family of over 6,000 T6SS effectors found in Gram-negative bacteria. My work on these proteins has uncovered that different regions found within effectors have distinct roles in trafficking between bacterial cells and in the growth inhibition of the target cell. / Dissertation / Doctor of Philosophy (PhD) / Bacteria constantly compete with their neighbours for resources and space. The type VI secretion system is a protein complex that facilitates competition between Gram-negative bacteria by facilitating the injection of protein toxins, also known as effectors, from attacking cells into target cells. In this work, I characterize several members of a large family of membrane protein effectors. First, I showed that these effectors require a novel family of chaperone proteins for stability and recruitment to the type VI secretion system apparatus. Second, I characterized the growth-inhibitory properties of one of these effectors in-depth and showed that it possesses a toxin domain that depletes the essential nucleotides ATP and ADP in target cells by synthesizing the nucleotides adenosine penta- and tetraphosphate, (p)ppApp. Together, these studies revealed a new mechanism for the intercellular delivery of membrane protein toxins and uncovered the first known physiological role of a (p)ppApp-synthesizing enzyme in bacteria.

Infectious and neurodegenerative diseases continue to be a major concern worldwide. In spite of the great advances in drug therapy for treating various infectious and neurodegenerative diseases, there is still an urgent need for new and improved drugs due to increasing drug resistance among pathogens, emergence of new pathogens, ease of transmission of infections, ineffective available treatments, toxicity associated with current standard of care, aging populations and the lack of better alternative treatment options. The first part of this manuscript (chapters 1 - 5) describes the synthesis of novel agents active against Leishmania donovani. According to the World Health Organization (WHO), a significant number of deaths worldwide can be attributed to infectious diseases – particularly neglected tropical diseases (NTDs), one of which is leishmaniasis - a complex and clinically diverse disease transmitted through the bite of an infected female phlebotomine sand-fly. The pathogen that causes leishmaniasis develops through a complex life cycle via different morphological changes. Its clinical presentations range from the less severe (cutaneous) to lethal/fatal (visceral) forms depending upon the level of systemic involvement, infecting species and the endemic environment. Treatments (and vaccines) must be species-specific to be particularly effective since sensitivity to commonly used drugs is largely species-specific. Heat shock protein 90 (Hsp 90) has been shown to promote the differentiation of the protozoan parasite that causes leishmaniasis from the promastigote stage to the amastigote pathogenic stages. To this end a series of compounds were prepared based on known Hsp 90 inhibitors, SNX2112 and XL888. The synthetic approach allows the probing of a hydrophobic pocket and rapid access to a collection of anti-leishmanial compounds. The most active compound, was found to be more than twice as active as the climivally used drug, miltefosine, in an infected J774 macrophage at IC50 = 0.65 µM. The second part of this manuscript (chapters 6 - 9) describes the synthesis novel anti-Alzheimer’s agents. Alzheimer’s disease is a progressive neurodegenerative disease believed to be caused by tau hyperphosphorylation and plaque aggregation in the brain. It is known to affect about 44 million people worldwide and it is marked as the 6th leading cause of death in the United States. Slingshot homology-1 (SSH1) proteins, important protein phosphatases, are promising targets for the discovery of a new generation of small molecule inhibitors as treatment for Alzheimer’s disease, since SSH1 is known to contribute to both tau hyperphosphorylation and plaque aggregation in the brain. Through structure and activity relationships (SAR) studies, two (2) series of compounds were synthesized, thiazoles and pyridones, bearing a carboxylic acid or phosphonic acid functionality as inhibitors of SSH1 enzymes. In the preliminary screening efforts against SSH1 phosphatase activity, the thiazole series were found to be more potent at inhibiting the phosphatase activity than the pyridone series. Among the active thiazole series, eight (8) analogs exhibited significant inhibitory activity over the initial hit compound, observed via phosphatase inhibition curves (using a pNPP phosphatase assay). Further investigations into the molecular target (SSH1) are currently underway.

Les chaperons moléculaires, une famille de protéines diverses en structure et taille, sont dédiés à accompagner, replier et protéger d’autres protéines afin qu’elles atteignent leur conformation finale et leur emplacement dans la cellule. Dans ce but, les chaperons moléculaires doivent être hautement spécialisés dans l’exécution de tâches spécifiques, telles que le repliement, le transport ou la désagrégation, et polyvalents dans leur motifs de reconnais- sance, afin de pouvoir interagir avec un grand nombre de protéines di érentes. Di érents chaperons moléculaires collaborent au sein de la cellule, formant ainsi un réseau complexe qui assure le contrôle de la qualité du protéome. Les interactions entre les di érents partenaires de ce réseau et entre les chap- erones et leurs substrats sont souvent dynamiques, ce qui rend leur obser- vation structurale particulièrement di cile pour les techniques de biologie structurale. Par conséquent, il y a à ce jour peu d’information sur les struc- tures et mécanismes d’interaction au sein des complexes chaperon-substrate. Dans cette thèse, je présente des études sur la structure, la dynamique et les interactions entre les substrats de deux chaperons moléculaires, en utilisant diverses méthodes biophysiques et in vivo.Dans la première partie, je montre que la chaperone TIM910, située dans l’espace inter-membranaire des mitochondries, lie ses substrats, des protéines membranaires destinées aux deux membranes mitochondriales, d’une manière très dynamique. Non seulement le complexe TIM910 est en échange constant entre les espèces monomèriques et hexameriques, mais aussi le substrat lié échange entre mulitples conformations à une échelle de millisecondes. Sur la base de la résonance magnétique nucléaire (RMN), de small-angle X-ray scat- tering (SAXS), de l’ultracentrifugation analytique (AUC) et des expériences mutationnelles in vivo et des tests fonctionnels d’import dans les mitochon- dries, je propose un modèle structurale de l’interaction entre le chaperon et la protéine membranaire. TIM910 lie ses substrats dans une poche hydrophobe à l’extérieur du chaperon. Cette interaction est modulaire et se fait avec un ou deux hexamères de TIM910, en fonction de la longueur du substrat.Dans la deuxième partie, nous avons étudié le comportement du récepteur N-terminal du unfoldase ClpC1 de M. tuberculosis en présence d’antibiotiques et de ligands di érents. Le domaine N-terminal de ClpC1 est le site de liai- son de divers antibiotiques nouveaux contre M. tuberculosis. L’antibiotique Cyclomarin A supprime complètement la dynamique induite par le ligand arginine-phosphate. Nous proposons que cette suppression de la dynamique soit le principe fondamental du mécanisme d’action de cet antibiotique.Dans les deux cas, les structures X-ray des chaperons dans leur état apo et la structure de ClpC-NTD liée à des antibiotiques étaient disponibles, mais ces structures statiques ne su sent pas pour expliquer le mécanisme d’action. La structure X-ray de TIM910 n’a pas fourni d’ indication sur l’endroit ou la façon dont les substrats sont liés. De même, les structures X-ray du domaine N-terminal de apo et de Cyclomarine A de ClpC1 ne présentent que des di érences de structure mineures. Les deux exemples montrent que les données structurelles statiques souvent ne permettent pas d’expliquer le fonctionnement d’un système moléculaire, donc la combinaison de di érentes techniques et le développement de nouvelles méthodes pour étudier les complexes chaperon-substrat sont primordiaux pour comprendre leur fonction. / The diverse group of molecular chaperones is dedicated to accompany, fold and protect other proteins until they reach their final conformation and loca- tion inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many di erent client pro- teins. Moreover, molecular chaperones need to be able to interact with each other and with other components of the protein quality control system in a complex network. Interactions between the di erent partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially di cult to study using standard structural biology tech- niques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. In this thesis I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods.In the first part I show that the mitochondrial membrane protein chap- erone TIM910 binds its substrates in a highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hex- americ species, but also the bound substrate samples multiple conformations on a millisecond timescale. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments I propose a structural model of the chap- erone/membrane protein interaction. TIM910 binds its substrates in a hy- drophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate.In the second part I studied the behavior of the N-terminal receptor do- main of the ClpC1 unfoldase from M.tuberculosis in the presence of di erent antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic.In both cases X-ray structures of the apo or antibiotic bound form were available, but not su cient to explain the mechanism of action. The X- ray structure of TIM910 provided no evidence on where or how substrates are bound. Likewise, X-ray structures of the apo and cyclomarin-bound N-terminal domain of ClpC1 show only minor di erences in structure.Both examples show that static structural data is often not enough to explain how a molecular system works, and only the combination of di er- ent techniques, including newly developed methods enable the atomic-level understanding of chaperone/substrate complexes.

Master of Science / Department of Human Nutrition / Denis M. Medeiros / Copper deficiency has been linked with many health problems, among them cardiac hypertrophy. Because of its potential for causing oxidative damage, copper within the cell must be bound to chaperone proteins. In this thesis, we examined the role of dietary copper deficiency in the regulation of select copper chaperone proteins in cardiac tissue of rats. Sixteen weanling male Long-Evans rats were randomized into treatment groups, one group receiving a copper deficient diet (< 1 mg Cu/kg diet) and one group receiving a diet containing adequate copper (6 mg Cu/kg diet) for 5 weeks. Rats were sacrificed and a small blood sample was removed to determine hematocrit. Also, heart and liver tissues were removed for subsequent analysis. Rats fed the copper deficient diet had lower body weights but greater heart weights and heart:body weight. Hematocrit levels and liver copper concentrations were markedly decreased in copper deficient rats. These variables indicated that the copper deficient diet did in fact induce a copper deficiency in these animals. Non-myofibrillar proteins from the hearts were removed and separated by SDS-PAGE. Western Blotting was used to determine the concentrations of CTR1, CCS, Cox17, SCO1, Cox1 and Cox4. No changes were observed in the concentrations of CTR1 and Cox17. CCS and SCO1 were up-regulated as a result of copper deficiency, while Cox1 and Cox4 were both down-regulated. However, use of another antibody against Cox subunits suggested that only the nuclear encoded subunits including subunit IV were decreased, but not subunits I and II. These data provide new insight into the cardiac hypertrophy observed in copper deficiency, which suggests that select chaperone proteins may be up-regulated by a dietary copper deficiency.

Many neurodegenerative diseases are characterized by the accumulation of misfolded proteins that often share common morphological and biochemical features, and can similarly co-localize with several other proteins, including various chaperone proteins. Chaperone proteins, like heat shock protein 27 (HSP27), heme oxygenase 1 (HO-1) and clusterin, have been implicated as potent modulators of misfolded proteins, thus may play important roles in the pathogenesis of neurodegenerative diseases. The present study aims to investigate their roles in the pathogenesis of Frontotemporal lobar degeneration (FTLD), Alzheimer's disease (AD), Parkinson's disease (PD), and Motor neuron disease (MND) by determining their distribution and amount via immunohistochemical staining and western blotting in diseased and control subjects.There were distinct patterns of HSP27 and clusterin immunostaining in different brain regions. For HSP27, patients with AD and FTLD were in general more severely affected than were patients with MND and control subjects. For clusterin, patients with AD and FTLD were more severely affected than control subjects where neurons and glial cells were concerned, while patients with AD and control subjects were more severely affected than those with FTLD where diffuse and cored plaques were concerned. However, there were no obvious differences in the pattern of HO-1 immunostaining in various brain regions in patients with AD or FTLD relative to control subjects. Moreover, there was no association between HSP27, HO-1 and clusterin with disease or histological type, and the ‘classic’ neuropathological changes in FTLD, AD and MND were not immunoreactive to any of these proteins. There were significant correlations between the degrees of HO-1 and clusterin immunostaining in many brain areas for both AD and FTLD cases, and for all cases overall, but none between HSP27 and clusterin or HSP27 and HO-1. Present results suggest an involvement with ongoing cellular stress, misfolded or unfolded protein accumulation or the deficits/failure of other relevant protein quality control systems, in the pathogenesis of these neurodegenerative diseases. Present work may therefore have implications for the further development of ideas concerning the cause or treatment of neurodegenerative diseases where there is aberrant accumulation of misfolded, aggregated protein, and perhaps for conformational diseases in general. However, there are still many issues remain to be elucidated. Further research aimed at understanding the function and mechanisms of the chaperone system, and other protein quality control mechanisms, in the pathogenesis of neurodegenerative diseases is still needed.

Molecular chaperones are proteins that interact with and aid in stabilization and activation of other proteins. Chaperones help proteins attain their three dimensional conformation, without forming a part of the final structure. Many of the chaperones are stress proteins known as Heat shock proteins (Hsps). Their expression is upregulated in response to various kinds of stress such as heat stress, oxidative stress etc., which threaten the protein homeostasis, by structurally destabilizing cellular proteins, and increasing the concentration of aggregation-prone folding intermediates. The Hsps are classified according to their molecular weight into Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp families. Some of them are constitutively expressed and play a fundamental role in de novo protein folding. They further aid in proteome maintenance by assisting in oligomeric assembly, protein trafficking, refolding of stress denatured protein, preventing protein aggregation and protein degradation. Heat shock protein 90 (Hsp90) are one of the important representatives of this class of proteins. Hsp90 are highly conserved class of molecular chaperones. They are found in bacteria, eukaryotes, but not in archaea. In contrast to the eukaryotes which require a functional cytoplasmic Hsp90 for viability, the bacterial counterpart (HtpG) is typically nonessential. Hsp90 is an ATP dependent chaperone. Hsp90 form dimers, with each protomer consisting of three functional domains: N- terminal, ATP binding domain, Middle domain and C-terminal domain. Hsp90 is a dynamic protein, and undergoes an elaborate conformational cycle during its ATPase cycle, which is essential for its chaperoning activity. The Hsp90 chaperone cycle is regulated by interaction with diverse cochaperones. Hsp90 interacts with specific set of substrate proteins. Many of these substrate proteins function at the heart of several cellular processes like signalling, cell cycle, apoptosis. Studies from protozoans like Leishmania, Plasmodium, Trypanosoma etc. have also implicated the role of Hsp90 in their growth and stage transitions. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. The ATP binding N-terminal domain (NTD), has been explored as the target domain for inhibition of Hsp90 using competitive inhibitors of ATP. Several chemical classes of Hsp90 inhibitors are known, including ansamycins, macrolides, purines, pyrazoles, and coumarin antibiotics. However, many inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs.inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs. The work presented in this thesis mainly concerns with the structural studies and biochemical and biophysical characterization of Hsp90 from two different sources viz. Dictyostelium discoideum, a cellular slime mould and a plant source Oryza sativa (rice). The structural analyses of these two proteins have been carried out by X-ray crystallography. Though yeast has been explored extensively as a model system to understand the different roles of Hsp90, it lacks the various signalling pathways essential for growth and development present in case of higher eukaryotes. D. discoideum has been employed as a model system to understand multicellular development, which occurs in response to starvation induced stress. D. discoideum has the advantages due to its ease of manipulation. The organism's genome also shows many signalling pathway for growth and differentiation that are conserved between D. discoideum and mammals. With this motivation, we have studied several structural aspects of the cytosolic isoform of Hsp90 from D. discoideum called HspD. HspD was also observed to play a role in the multicellular development of D. discoideum. It has been demonstrated that the treatment of D. discoideum with inhibitors like Geldanamycin or Radicicol causes an arrest in the multicellular development at the mound stage, and the few which escaped this arrest gave rise to abnormal fruiting bodies. A subset of the proteins involved in this mound arrest phenotype, were observed to have homologs in humans, which are clients of Hsp90. Therefore, a structural perspective of HspD can aid in better understanding of the role of this protein in the organism, as well as, elucidate any structural differences observed as compared to other species, which may have an impact on its activity. Studies on the physiological role of Hsp90 in plants began much later as compared to fungi and humans. In plants Hsp90 are involved in various abiotic stress responses. In addition, their roles have also been implicated in plant growth and development, innate immune response and buffering genetic variations. However, the molecular mechanisms of these various actions are not clearly understood. Also, the structural aspects of plant Hsp90 are yet to be explored. The structure of the NTD of Hsp90 from barley is the only one available from a plant source till now. We have initiated the studies on rice Hsp90 with the objective to understand the mechanism of Hsp90 in plants, which may aid in improving stress tolerance in plants. The thesis has been divided into five chapters. The first chapter introduces the various aspects of Hsp90 protein. The chapter starts with a general overview of concept of molecular chaperones and describes briefly the different classes of molecular chaperones. This is followed by a detailed description of different aspects of Hsp90 with main emphasis on the structure and its conformational flexibility. The chapter describes the association of Hsp90 with other accessory proteins like cochaperones and its interaction with its substrate proteins and explains the functional significance of Hsp90 as a drug target and the need for the development of new class of inhibitors, followed by the significance of the study of Hsp90 in the two model systems (D. discoideum and rice) chosen to be studied. The second chapter gives a brief overview of the principles behind the different experimental methods employed during the course of this research, which includes the tools of X-ray crystallography and other biochemical and biophysical techniques employed for the characterization of the protein. Chapter 3 describes the crystal structure of NTD of Hsp90 from D. discoideum. The structure of NTD was solved in two different native (ligand-free) forms viz. monoclinic and hexagonal. The two forms differed in local structural rearrangement of a segment of NTD known as the lid region. The lid region in the hexagonal form showed a shift in its position as compared to the other solved structures of NTD. The structure of NTD was also solved in complex with various ligands which include ADP, substrate analogs and an inhibitor molecule. A comparison of all the structures showed that the overall structure is well-conserved. One of the crystal structures of NTD showed a heptapeptide (part of the vector) bound at the active site. The peptide was observed to make several complementary interactions with the residues of the ATP binding pocket and retain several interactions which the nucleotide makes with the NTD. The NTD showed subtle conformational differences when compared with the NTD of Hsp90 from yeast. Chapter 4 details the structural and functional characteristics of full length Hsp90 protein from D. discoideum. Due to the large size and flexibility, the full length protein did not crystallize in spite of several attempts. Hence, HspD was studied using different solution studies like Small Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS). Both the studies showed the presence of higher oligomers. The SAXS data showed the presence of tetramers and hexamers while, the addition of the ligand shifts the protein from a dimer to a higher oligomer as observed from DLS studies. The chapter also describes the study of interaction of HspD with a cochaperone protein p23. The interactions were studied using ITC, which showed a strong binding. The ATPase activity was also evaluated in the presence of increasing concentrations of p23, which was observed to decline with increasing concentrations of p23. In chapter 5, we describe the biochemical characterization of Hsp90 from Oryza sativa (rice) and the crystallographic analysis of its NTD. Binding of the rice Hsp90 to ATP and an inhibitor were studied by fluorescence. The ATPase activity of rice Hsp90 was checked by radioactive assay and the protein was observed to be active. The NTD of rice Hsp90 crystallized as a monomer in complex with a substrate analog AMPPCP and the structure was determined.