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Neurodegenerative diseases are frequently characterised by the build-up of misfolded proteins and degeneration of brain structures. Tauopathies are a collective of >20 such diseases featuring abnormally aggregating tau, a microtubule-associated protein normally acting to stabilise these protein cargo tracks. There is currently no cure. Small heat shock proteins (sHSPs) are highly conserved molecular chaperones known to act as holding partners for substrates. Studies have reported that some sHSPs can act as chaperones for disease-related proteins to prevent their aggregation and may be beneficial in such diseases. However, there is a lack of a thorough screen of the sHSPs and their disease-modulating effects in an in vivo model. The aim of this thesis research was to screen the sHSP family to determine which, and how, any of the human sHSPs could ameliorate tau toxicity in zebrafish tauopathy models. Of all eleven sHSPs, HSPB1, B4 and B5 ameliorated morphological disease phenotypes induced by pan-neuronal expression of mutant A152T tau. HSPB4 and B5 have never been demonstrated to be beneficial in a tauopathy model in vivo, and peptides created from the central domain of these similarly ameliorated the morphological phenotype. To further investigate the underlying mechanism of action, I created and characterised a transgenic zebrafish line ubiquitously expressing human HSPB5. I demonstrate that overexpression of HSPB5 in the pan-neuronal A152T tau line results in reduced levels of hyperphosphorylated and insoluble tau. Additionally, a synthetic HSPB5 peptide similarly ameliorated morphological phenotypes by treatment via embryo immersion and reduced levels of insoluble tau. These results indicate that HSPB5 and its peptide may be of therapeutic interest for use against tauopathies as it can improve various disease phenotypes in an in vivo model.

The folding of proteins into their native structure is crucial for the functioning of all biological processes. Molecular chaperones are guardians of the proteome that assist in protein folding and act to prevent the accumulation of aberrant protein conformations that can lead to proteotoxicity. Despite their important functions in protein folding homeostasis, it is not entirely clear the underlying mechanisms of how chaperones assist in protein folding. This thesis focused on understanding the mechanism of the specific class of chaperones that do not use ATP or other cofactors to regulate their chaperone activity. These ATP-independent chaperones are generally assumed to act as “holdase” chaperones that work by tightly holding onto non-native proteins and thus preventing them from aggregating. This traditional “holdase” perspective that ATP-independent chaperones are not directly involved in the folding process had been recently challenged. Our recent studies using the periplasmic chaperone Spy illustrates that client proteins can explore different conformational states while bound to this ATP-independent chaperone. This thesis aims to (1) understand the principles underlying how proteins can fold while they remain continuously bound to a chaperone, and (2) examine whether this folding-while-bound mechanism is utilized by other classic “holdase” chaperones. In the first part of this thesis, super Spy variants (H96L and Q100L) were used to show that strengthening the interaction between Spy and its client protein strongly inhibits the ability of the client to fold while chaperone bound. The results showed that more tightly Spy binds to its clients, the more it slows the folding rate of the bound client. Efficient chaperone-mediated folding while bound appears to represent an evolutionary balance between interactions of sufficient strength to mediate folding and interactions that are too tight, which tend to inhibit protein folding. In the second part of this thesis, trigger factor was used as another ATP-independent chaperone to test if allowing proteins to explore different conformational states while chaperone bound could be an underlying principle that also applies to other chaperones that have been thought to act as simple “holdases”. Trigger factor rapidly binds to partially folded glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and prevents it from non-productive self-association by shielding oligomeric interfaces from aggregation, consistent with a role as an aggregation inhibiting holdase chaperone. Surprisingly, non-native GAPDH folds into a monomeric but otherwise rather native-like intermediate state while trigger factor-bound. Upon release from trigger factor, this mostly folded monomeric GAPDH rapidly self-associates and acquires enzymatic activity. The work presented here shows how chaperones can bridge the holdase-foldase divide and demonstrates how an ATP-independent chaperone can be actively involved in the protein folding process.

Maintaining protein homeostasis is fundamental for organismal survival. Integral to this process are molecular chaperones, including the highly conserved heat shock protein 90 (Hsp90). Hsp90 associates with many ‘client’ proteins throughout their lifetime, regulating the transition from partially unfolded, inactive states to native, active states. However, a mechanistic understanding of how Hsp90 remodels and regulates client proteins is lacking. One class of clinically important Hsp90 clients are the steroid hormone receptors (SHRs), including the model Hsp90 client, the glucocorticoid receptor (GR). GR is a ligand-activated transcription factor that constantly relies on Hsp90 to maintain its activity. In addition to facilitating folding of GR, Hsp90 regulates multiple steps in the GR activation pathway, including ligand binding, post-translation modifications (PTMs), nuclear translocation, and DNA binding. To perform these regulatory functions, Hsp90 requires the molecular chaperone Hsp70 as well as a collection of cochaperones (p23, Hop, FKBP51, FKBP52, PP5, and others). Using cryo-EM, as well as biochemical and biophysical techniques, we investigated the molecular mechanisms describing how Hsp90, Hsp70, and various cochaperones modify the conformational state of GR and regulate various steps in the GR activation pathway. We present two cryo-EM structures that reveal, for the first time, the mechanism of how Hsp90 folds a client protein. We present the GR-loading complex (GR:Hsp90:Hsp70:Hop), in which GR is stabilized in an inactive, partially unfolded conformation that cannot bind ligand, and the GR-maturation complex (GR:Hsp90:p23), in which GR is restored to a folded, ligand-bound, active state. Together, our structures reveal how Hsp70 and Hsp90 cooperate to regulate the conformational state of a client to directly control the client’s ability to bind ligand for activation. We also present three additional cryo-EM structures (GR:Hsp90:FKBP51, GR:Hsp90:FKBP52, and GR:Hsp90:PP5), which demonstrate how Hsp90 acts a regulatory platform to couple the conformational state of the client protein to distinct client fates through interactions with various cochaperones.

Molecular chaperones bind to misfolded proteins (“clients”) to protect them from aggregation and promote their folding. The following Chapters all focus on understanding how the protein-protein interactions (PPIs) between members of the molecular chaperone network and their client proteins regulate these processes. Specifically, I develop a functional genomic approach to explore which molecular chaperones are required for the stability of specific client proteins (Chapter 2) and use biophysical and biochemical approaches to probe how conserved amino acid sequences in the heat shock protein 70 (Hsp70) chaperone allow it to engage with two different classes of co-chaperones (Chapter 3). Together, these studies and recent literature reports suggest that weak interactions (i.e. micromolar) are critical to the molecular logic of chaperone-mediated folding.

Heat shock protein 70 (Hsp70) is an abundant and ubiquitous molecular chaperone that is responsible for maintenance of the human proteome. Accordingly, Hsp70 has become an attractive drug target for neurodegenerative and hyperproliferative disorders; however it is difficult to imagine strategies for inhibiting its pathobiology without impacting its essential roles. Fortunately, Hsp70 does not work alone, and instead employs a large network of co-chaperone proteins, which can tune Hsp70 activity and influence disease state. These co-chaperone proteins provide potential handles for targeting Hsp70 without disrupting overall proteostasis. One such class of co-chaperones proteins known as the Nucleotide Exchange Factors (NEFs), are a particular appealing target. NEFs bind Hsp70 and help to facilitate the exchange of ADP for ATP. The biochemistry of the NEF family of co-chaperones has classically been investigated using the prokaryotic NEF, GrpE, as a model. However, the eukaryotic cytosol does not contain a GrpE homolog. Rather, there are three main sub-classes of human NEFs: Hsp110, HspBP1, and the BAG proteins, all of which are structurally distinct with little sequence homology. Consistent with their diverse structures, they also differ in their mode of binding to Hsp70 and their roles in guiding Hsp70 biology. For example, BAG2 is associated with proteasomal degradation of the Hsp70 substrate, tau, while BAG1-Hsp70 is linked to increased tau stability. These observations suggest that the formation of specific NEF-Hsp70 complexes may help decide the fate of Hsp70-bound substrates. Additionally, these findings illustrate that differential disruption of specific Hsp70-NEF contacts might be beneficial in disease. In this thesis work I have systematically characterized the human Hsp70 NEFs, including how they interact with Hsp70, how the influence Hsp70 biochemistry and how they can bridge Hsp70 with other classes of chaperone proteins. I have used high throughput screening methods to search for chemical matter that can modulate Hsp70-NEF interactions, and we have shown that inhibitors of Hsp70-NEF interactions can be beneficial for treating disease. This thesis work has significantly advanced our knowledge of human Hsp70 regulation, and has provided groundwork for future studies on other Hsp70 co-chaperones and proteostasis components.

Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes ranging from cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the last decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, a large number of both permanent and transient RP components with specialized functional roles are critical for proteasome function. This research investigates the dynamic nature of protein-protein interactions involved in proteasome assembly and substrate recruitment, and how they provide context to our current understanding of proteasome activation by the RP.

Historically, peptidases have been considered as housekeeping proteins involved in protein degradation and amino acid turnover. However, recent work has highlighted the fact that peptidases, including aminopeptidases, have critical regulatory roles in the cell including modulation of growth, development and stress responses. Recently, plant leucine aminopeptidases (LAPs) have been recognized for their roles in modulation of late wound-responses and insect defense. While no known mechanism of action has been identified, LAPs were presumed to act through their aminopeptidase function to affect the stability, activity, or localization of a peptide and protein involved in defense. In my dissertation studies, I have identified two new molecular functions for plant LAPs as molecular chaperones and Cys-Gly dipeptidases. In addition, microarray analyses has demonstrated the tomato LAP-A regulates early and late wound responses both positively and negatively. In particular, microarray analyses identified two new sets of genes modulated by LAP-A: late wound dehydrins and Pathogenesis-Related 1. This study also provides evidence LAP-A may act though the negative regulator salicylic acid or the positive regulator hydrogen peroxide to modulate wound signaling. The role of LAP-A in glutathione metabolism is also discussed. Together this study has provided evidence that LAP-A's role in wound signaling and insect defense is more complex than initially anticipated and may be the result of LAP-A utilizing multiple functions (aminopeptidase, chaperone and/or Cys-Gly dipeptidase activities) in different environments and in response to different stresses.

Tauopathies are a class of neurodegenerative disorders, which affect millions of people worldwide. These diseases are characterized by the accumulation of protein aggregates composed of abnormally modified variants of the microtubule-binding protein tau. Despite extensive efforts, there is currently no cure for these devastating diseases. The goal of this study is to understand whether heat shock protein 70 (Hsp70) may be an under-explored target for tauopathies. Hsp70 is a molecular chaperone that plays important roles in protein quality control, including the turnover of misfolded polypeptides. However, the molecular mechanisms of tau quality control were not known and it wasn’t clear how pharmacological inhibition of Hsp70 might impact tau accumulation. In this thesis, we review the literature linking Hsp70 to tau and hypothesize that inhibiting the ATPase activity of Hsp70 might lead to the degradation of aberrant tau by favoring the triage pathway. To test this hypothesis, we developed a new high-throughput screen platform and identified promising inhibitors, including methylene blue (MB). MB inhibited the ATPase activity of Hsp70 and, consistent with the major hypothesis, caused a dramatic, Hsp70-dependent reduction in the accumulation of tau in cellular and animal models of tauopathy. In mechanistic studies, we found that MB inhibits Hsp70 by selectively oxidizing two key cysteine residues in the chaperone. To further explore this process, we developed a parallel chemical series of Hsp70 inhibitors based on the known anti-cancer agent, MKT-077. We found that these compounds inhibit the ATPase activity of Hsp70 and, like MB, they dramatically reduce tau levels. However, the MKT-077 analogs acted by a distinct mechanism: binding to the ADP-bound form of Hsp70 and allosterically blocking nucleotide cycling. Together, these studies suggest that Hsp70 normally protects tau from degradation, while inhibitors lead to its rapid degradation. In summary, we have used a chemical biology approach to show that Hsp70 plays a critical role in tau stability and inhibitors reduce tau levels. Thus, this work suggests a potential path towards new treatments for tauopathies. In addition, these findings provide molecular insights into how Hsp70 mediates protein quality control.

The molecular chaperone, heat shock protein 70 (Hsp70), is an important regulator of protein homeostasis that has roles in both primary protein folding and clearance of misfolded proteins. Several studies have suggested that it is the interaction between Hsp70 and a network of co-chaperones that might determine the fate Hsp70-bound substrates. For example, a complex formed between Hsp70 and the E3 ubiquitin ligase CHIP, is thought to guide substrates to the proteasome. However the mechanism governing how co-chaperones assemble on Hsp70 remains unresolved. We wanted to explore how interactions between Hsp70 and CHIP govern the ubiquitin-mediated degradation of chaperone substrates. Moreover, we hypothesize that chemical disruption of the Hsp70- CHIP contact might decrease turnover of these substrates and, thereby, alter protein homeostasis. This thesis work involves the biophysical and structural characterization of the Hsp70-CHIP complex and synthesis of chemical probes that block CHIP binding to Hsp70. Using truncations and biochemical assays, we determined that the xvi tetratricopeptide repeat (TPR) domain of CHIP, along with an important secondary interaction in the charged coiled-coil domain govern binding of CHIP to Hsp70. In addition, we developed a dramatically improved synthesis of the promising natural product, spergualin, and created a collection of more potent and stable derivatives. We then showed that these compounds disrupt the Hsp70-CHIP interaction in vitro and in cells. Together, these findings provide a quantitative exploration of the CHIP-Hsp70 complex and the first set of chemical tools that can be used to regulate Hsp70 interactions with CHIP. We expect that these probes will be useful in further studying Hsp70-mediated protein quality control.

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone that makes up 1-2% of all cytosolic proteins under normal conditions and increases to 4-6% under stress. Unlike other members of the chaperone family, Hsp90 interacts in the later stages of protein folding promoting subtle rearrangements and client protein activation. Although no substrates have been confirmed to date for the bacterial homolog, the eukaryotic homologs have been shown to interact with a wide variety of proteins including kinases, steroid hormone receptors, nitric oxide synthase, and telomerase. Given the critical nature of the proteins listed, Hsp90 has been the focus of several cancer studies and become an attractive target for therapeutics. Crystal structures of the bacterial and yeast Hsp90 homologs provided the first major insight into the role of nucleotide and the dramatic conformational changes that Hsp90 undergoes during its nucleotide cycle. Electron microscopy and SAXS studies further examined these conformational changes and revealed that unlike other chaperones, Hsp90 does not undergo discrete nucleotide driven conformational changes but rather displays a dynamic conformational equilibrium of multiple states that is then shifted towards one state or another by nucleotide. Using ATP hydrolysis and mutational analysis I investigated the interplay between ATP binding and hydrolysis on the closed conformation of Hsp90. Residues of both monomers were shown to participate in a cross-monomer hydrophobic interaction network that synergistically stabilizes a hydrolysis competent conformation providing the first explanation for structural cooperativity and the importance of dimer formation. While investigating these interactions I was also able to clarify the role of the Middle Domain arginine by showing that it is directly involved in stabilizing the closed state of Hsp90 and not involved in the catalysis of ATP as previously thought. All of these studies were built upon the successes of solving crystal structures of Hsp90 homologs in the apo and nucleotide bound states. A large portion of my work was also focused on utilizing other homologs, nucleotide analogs, small molecules, co-chaperones and substrates to attempt to crystallize a novel conformation of Hsp90 that might reveal new insights into its chaperoning mechanism.

How iron-sulfur clusters are syntheisized in vivo remains an interesting topic to explore. In this dissertation, studies were focused on human NFU, an important protein that participates in iron-sulfur cluster biosynthesis. Studies were first carried out on NFU’s functional characterization. Due to the presence of the conserved CXXC motif in NFU, this protein was proposed and demonstrated to cleave the persulfide bond on NifS. In the presence of L-cysteine, a catalytic amount of NifS, ferrous iron and NFU, apo proteins lacking iron-sulfur clusters can be successfully reconstituted. The function of human NFU led to the hypothesis that this protein can interact with NifS. The binding of NFU to NifS was quantified by ITC (isothermal titration calorimetry). The temperature-dependence of binding was also examined to determine the underlying thermodynamics. A positive change in enthalpy with increased temperature suggested dominance of polar charged residues in the binding. The interaction of human NFU and the chaperone system was also studied in vitro. Both NFU and its C-terminal domain can stimulate the ATPase activity of the chaperone HscA. The ADP form of HscA binds to both NFU and the C-terminal domain more tightly than the ATP form, providing additional evidence for NFU interacting with the chaperone system. The binding of NFU to the HscA/HscB complex is tighter than binding to HscA alone, which is consistent with previous ATPase results. Besides functional characterization, structural characterization was also carried out. The near UV CD data for NFU were almost negligible, suggesting a possible molten globule property of human NFU. Results from ANS binding and tryptic digestion experiments were consistent with molten globule type proteins. Tryptic digestion suggested the rigid tertiary structure of the N-terminal domain. It was confirmed to possess a tertiary structure by the ANS experiment and the tryptic digestion experiment.

The redox-regulated molecular chaperone Hsp33 senses oxidative stress through oxidation of the four invariant cysteines of its zinc center. The zinc center occupies the carboxyl-terminal domain of Hsp33. Reactive oxygen species induce the formation of disulfide bonds connecting the nearby cysteines in CXC and CXXC motifs. It has been previously shown that the activation of Hsp33 by hydrogen peroxide is highly temperature-dependent. I report here that elevated temperatures cause the unfolding of the C-terminal regulatory domain. This domain unfolding exposes the substrate-binding site and enables Hsp33 to dimerize into its highly active form. Hsp33 is one of only a few proteins that have been shown to require partial unfolding to gain activity. Oxidative stress-induced disulfide bond formation locks Hsp33 in this partially unfolded, active state. This maintains Hsp33 in its active conformation until reducing, non-stress conditions are restored. An alternative activator of Hsp33 is the antimicrobial oxidant hypochlorous acid. Hypochlorous acid is produced by neutrophils as part of the immune response and by epithelial tissue lining the bronchi and the gut to deter colonization by microbes. In contrast to peroxide, hypochlorous acid causes the unfolding of numerous proteins. I found that while most proteins are inactivated by hypochlorous acid, Hsp33 becomes activated. This hypochlorous acid-induced unfolding of Hsp33 alleviates the need for elevated temperatures for activation by peroxide. It therefore appears that Hsp33 uses the unfolding activity of hypochlorous acid to stimulate its own chaperone activity. Hsp33 is activated by the very conditions that lead to protein unfolding and aggregation inside the cell, ensuring that Hsp33 is activated only when needed. This effectively protects bacteria against these oxidative stress conditions. In fact, other chaperones have been shown to lose their activity under these oxidizing, unfolding conditions. Thus, it is no surprise that nearly all bacteria possess the powerful chaperone Hsp33.