Characterization of NARJ: A molecular chaperone involved in assembly of nitrate reductase

The major goal of this work was to define the role of accessory protein, NARJ, in assembly of nitrate reductase which is a membrane-bound multisubunit enzyme that can catalyze the reduction of nitrate to nitrite under anaerobic growth in E. coli. Nitrate reductase is encoded by the nar GHJI operon under control of the narG promoter. The purified nitrate reductase is composed of three subunits: $\alpha,\ \beta,$ and $\gamma.$ The NARJ protein which is encoded by the third gene (narJ) is not found to be associated with any of the purified preparations of the enzyme, but is required for active nitrate reductase. In this study the product of the narJ gene was identified. NARJ appeared to be produced at a reduced level, compared to the other proteins encoded by the nar operon. Since NARJ could not be overexpressed to a level for an efficient purification, NARJ was expressed and purified as a recombinant protein with polyhistidine tag. The recombinant protein NARJ-6His could functionally replace native NARJ. Purified NARJ-6His is a dimeric protein which contains no identifiable cofactors or unique secondary structure. NARJ was localized in the cytoplasm, and was not associated with nitrate reductase in the membrane. In vivo NARJ activated the $\alpha\beta$ complex and stabilized the $\alpha$ subunit against protease degradation. In the absence of the membrane-bound $\gamma$ subunit, NARJ formed an intermediate complex with $\alpha\beta$ in the cytosol. Based on these studies, NARJ fits the formal definition of a molecular chaperone. It appears to be required only for the biogenesis of nitrate reductase and, therefore, is defined as a private chaperone specifically involved in the assembly of nitrate reductase system. ^

Biochemical and genetic characterization of the Saccharomyces cerevisiae Hsp110 molecular chaperone Sse1

The Ssel/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a carboxyl-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an amino-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Δ phenotypes. Surprisingly, all mutants predicted to abolish ATP hydrolysis complemented the temperature sensitivity of sse1Δ, whereas mutations in predicted ATP binding residues were non-functional. Remarkably, the two domains of Ssel when expressed in trans functionally complement the sse1Δ growth phenotype and interact by coimmunoprecipitation analysis, indicative of a novel type of interdomain communication. ^ Relatively little is known regarding the interactions and cellular functions of Ssel. Through co-immunoprecipitation analysis, we found that Ssel forms heterodimeric complexes with the abundant cytosolic Hsp70s Ssa and Ssb in vivo. Furthermore, these complexes can be efficiently reconstituted in vitro using purified proteins. The ATPase domains of Ssel and the Hsp70s were found to be critical for interaction as inactivating point mutations severely reduced interaction efficiency. Ssel stimulated Ssal ATPase activity synergistically with the co-chaperone Ydj1 via a novel nucleotide exchange activity. Furthermore, FES1, another Ssa nucleotide exchange factor, can functionally substitute for SSE1/2 when overexpressed, suggesting that Hsp70 nucleotide exchange is the fundamental role of the Sse proteins in yeast, and by extension, the Hsp110 homologs in mammals. ^ Cells lacking SSE1 were found to accumulate prepro-α-factor, but not the cotranslationally imported protein Kar2, similar to mutants in the Ssa chaperones. This indicates that the interaction between Ssel and Ssa is functionally significant in vivo. In addition, sse10 cells are compromised for cell wall strength, likely a result of decreased Hsp90 chaperone activity with the cell integrity MAP kinase SIC. Taken together, this work established that the Hsp110 family must be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.^

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1.

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.