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

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

Feasibility of the Transport PLUS Intervention to Improve the Transitions of Care for Patients Transported Home by Ambulance: a Non-Randomized Pilot Study

BACKGROUND: The growing population of patients over the age of 65 faces particular vulnerability following discharge after hospitalization or an emergency room visit. Specific areas of concern include a high risk for falls and poor comprehension of discharge instructions. Emergency medical technicians (EMTs), who frequently transport these patients home from the hospital, are uniquely positioned to aid in mitigating transition of care risks and are both trained and utilized to do so using the Transport PLUS intervention. METHODS: Existing literature and focus groups of various stakeholders were utilized to develop two checklists: the fall safety assessment (FSA) and the discharge comprehension assessment (DCA). EMTs were trained to administer the intervention to eligible patients in the geriatric population. Using data from the checklists, follow-up phone calls, and electronic health records, we measured the presence of hazards, removal of hazards, the presence of discharge comprehension issues, and correction or reinforcement of comprehension. These results were validated during home visits by community health workers (CHWs). Feasibility outcomes included patient acceptance of the Transport PLUS intervention and accuracy of the EMT assessment. Qualitative feedback via focus groups was also obtained. Clinical outcomes measured included 3-day and 30-day readmission or ED revisit. RESULTS: One-hundred three EMTs were trained to administer the intervention and participated in 439 patient encounters. The intervention was determined to be feasible, and patients were highly amenable to the intervention, as evidenced by a 92% and 74% acceptance rate of the DCA and FSA, respectively. The majority of patients also reported that they found the intervention helpful (90%) and self-reported removing 40% of fall hazards; 85% of such changes were validated by CHWs. Readmission/revisit rates are also reported. CONCLUSIONS: The Transport PLUS intervention is a feasible, easily implemented tool in preventative community paramedicine with high levels of patient acceptance. Further study is merited to determine the effectiveness of the intervention in reducing rates of readmission or revisit. A randomized control trial has since begun utilizing the knowledge gained within this study

First virtual international congress on cellular and organismal stress responses, november 5–6, 2020

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5–6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI

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

Heat shock in the springtime

International audienceA collaborative workshop dedicated to the discussion of heat shock factors in stress response, development, and disease was held on April 22-24, 2014 at the Université Paris Diderot in Paris, France. Recent years have witnessed an explosion of interest in these highly conserved transcription factors, with biological roles ranging from environmental sensing to human development and cancer