Oxidation of Two Cysteines Within Yeast HSP70 Impairs Proteostasis While Directly Triggering an HSF1-Dependent Cytoprotective Response

Neurodegenerative diseases such as Alzheimer\u27s, Parkinson\u27s, and Huntington\u27s diseases affect millions of Americans every year. One factor linked to the formation of aggregates associated with these diseases is damage sustained to proteins by oxidative stress. Management of protein misfolding by the ubiquitous Hsp70 chaperone family can be modulated by modification of two key cysteines in the ATPase domain by oxidizing or thiol-modifying compounds. To investigate the biological consequences of cysteine modification on the Hsp70 Ssa1 in budding yeast, we generated cysteine null (cysteine to serine) and oxidomimetic (cysteine to aspartic acid) mutant variants of both C264 and C303 and demonstrate reduced ATP binding, hydrolysis, and protein folding properties in both the oxidomimetic and hydrogen peroxide-treated Ssa1. In contrast, cysteine nullification rendered Ssa1 insensitive to oxidative inhibition. Additionally, we determined the oxidomimetic ssa1-2CD (C264D, C303D) allele was unable to function as the sole Ssa1 isoform in yeast cells and also exhibited dominant negative effects on cell growth and viability. Ssa1 binds to and represses Hsf1, the major transcription factor controlling the heat shock response, and we found the oxidomimetic Ssa1 failed to stably interact with Hsf1, resulting in constitutive activation of the heat shock response. Consistent with our in vitro findings, ssa1-2CD cells were compromised for de novo folding, post-stress protein refolding, and in regulated degradation of a model terminally misfolded protein. Together, these findings pinpoint Hsp70 as a key link between oxidative stress and proteostasis, information critical to understanding cytoprotective systems that prevent and manage cellular insults underlying complex disease states

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

Cytoplasmic Redox Imbalance in the Thioredoxin System Activates Hsf1 and Results in Hyperaccumulation of the Sequestrase Hsp42 With Misfolded Proteins

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH, and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a process of long-term sequestration

Quantitative Proteomic Analysis Reveals Unique HSP90 Cycle-Dependent Client Interactions

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states

Structure of the Hsp110:Hsc70 nucleotide exchange machine.

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein\u27s NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs

Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker

A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells

Making Connections - Envisioning Springfield\u27s North End

This work explores a service learning strategy in the context of the senior Urban Design Studio taught in the Department of Landscape Architecture and Regional Planning at the University of Massachusetts Amherst. The primary goal of this project is to stimulate a conversation in the neighborhoods of the North End, to develop green design strategies, to improve services and businesses for residents and the employees of local businesses, and to foster cultural engagement and interaction in the North End that will enhance the vibrancy, resilience, and quality of life of this urban community. Making connections - Envisioning Springfield\u27s North End proposes improved connectivity in a physical, cultural, and social sense will be key to attaining these goals and to engaging and synergizing individuals and community groups in the North End - residents, businesses, schools, churches, employers, and employees. Six sustainable learning and planning principles have emerged from this studio: 1. Input and interaction – Visioning workshops connect campus and community 2. Community-building art - Expression of place and people 3. Healthy living - Urban agriculture and education 4. Urban greenways – Abandoned railways and urban rivers and streams 5. Green infrastructure - Green streets as networks and structural framework 6. Sustainable urban form – Mixed use and pedestrian friendly neighborhood

SYM1 Is the Stress-Induced Saccharomyces cerevisiae Ortholog of the Mammalian Kidney Disease Gene Mpv17 and Is Required for Ethanol Metabolism and Tolerance during Heat Shock

Organisms rapidly adapt to severe environmental stress by inducing the expression of a wide array of heat shock proteins as part of a larger cellular response program. We have used a genomics approach to identify novel heat shock-induced genes in Saccharomyces cerevisiae. The uncharacterized open reading frame (ORF) YLR251W was found to be required for both metabolism and tolerance of ethanol during heat shock. YLR251W has significant homology to the mammalian peroxisomal membrane protein Mpv17, and Mpv17(−/−) mice exhibit age-onset glomerulosclerosis, deafness, hypertension, and, ultimately, death by renal failure. Expression of Mpv17 in ylr251wΔ cells complements the 37°C ethanol growth defect, suggesting that these proteins are functional orthologs. We have therefore renamed ORF YLR251W as SYM1 (for “stress-inducible yeast Mpv17”). In contrast to the peroxisomal localization of Mpv17, we find that Sym1 is an integral membrane protein of the inner mitochondrial membrane. In addition, transcriptional profiling of sym1Δ cells uncovered changes in gene expression, including dysregulation of a number of ethanol-repressed genes, exclusively at 37°C relative to wild-type results. Together, these data suggest an important metabolic role for Sym1 in mitochondrial function during heat shock. Furthermore, this study establishes Sym1 as a potential model for understanding the role of Mpv17 in kidney disease and cardiovascular biology