The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis

Hsp90, a dimeric ATP‐dependent molecular chaperone, is required for the folding and activation of numerous essential substrate “client” proteins including nuclear receptors, cell cycle kinases, and telomerase. Fundamental to its mechanism is an ensemble of dramatically different conformational states that result from nucleotide binding and hydrolysis and distinct sets of interdomain interactions. Previous structural and biochemical work identified a conserved arginine residue (R380 in yeast) in the Hsp90 middle domain (MD) that is required for wild type hydrolysis activity in yeast, and hence proposed to be a catalytic residue. As part of our investigations on the origins of species‐specific differences in Hsp90 conformational dynamics we probed the role of this MD arginine in bacterial, yeast, and human Hsp90s using a combination of structural and functional approaches. While the R380A mutation compromised ATPase activity in all three homologs, the impact on ATPase activity was both variable and much more modest (2–7 fold) than the mutation of an active site glutamate (40 fold) known to be required for hydrolysis. Single particle electron microscopy and small‐angle X‐ray scattering revealed that, for all Hsp90s, mutation of this arginine abrogated the ability to form the closed “ATP” conformational state in response to AMPPNP binding. Taken together with previous mutagenesis data exploring intra‐ and intermonomer interactions, these new data suggest that R380 does not directly participate in the hydrolysis reaction as a catalytic residue, but instead acts as an ATP‐sensor to stabilize an NTD‐MD conformation required for efficient ATP hydrolysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92411/1/2103_ftp.pd

Shaping the Future of Research: a perspective from junior scientists

The landscape of scientific research and funding is in flux as a result of tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2 nd and 3 rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes

Outcomes of the “BRCA Quality Improvement Dissemination Program”: An Initiative to Improve Patient Receipt of Cancer Genetics Services at Five Health Systems

OBJECTIVE: A quality improvement initiative (QII) was conducted with five community-based health systems\u27 oncology care centers (sites A-E). The QII aimed to increase referrals, genetic counseling (GC), and germline genetic testing (GT) for patients with ovarian cancer (OC) and triple-negative breast cancer (TNBC). METHODS: QII activities occurred at sites over several years, all concluding by December 2020. Medical records of patients with OC and TNBC were reviewed, and rates of referral, GC, and GT of patients diagnosed during the 2 years before the QII were compared to those diagnosed during the QII. Outcomes were analyzed using descriptive statistics, two-sample t-test, chi-squared/Fisher\u27s exact test, and logistic regression. RESULTS: For patients with OC, improvement was observed in the rate of referral (from 70% to 79%), GC (from 44% to 61%), GT (from 54% to 62%) and decreased time from diagnosis to GC and GT. For patients with TNBC, increased rates of referral (from 90% to 92%), GC (from 68% to 72%) and GT (81% to 86%) were observed. Effective interventions streamlined GC scheduling and standardized referral processes. CONCLUSION: A multi-year QII increased patient referral and uptake of recommended genetics services across five unique community-based oncology care settings

A call for transparency in tracking student and postdoc career outcomes

There is a common misconception that the United States is suffering from a “STEM shortage,” a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes

A call for transparency in tracking student and postdoc career outcomes

There is a common misconception that the United States is suffering from a “STEM shortage,” a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes

Basal Activity of a PARP1-NuA4 Complex Varies Dramatically across Cancer Cell Lines

Poly(ADP-ribose) polymerases (PARPs) catalyze poly(ADP-ribose) addition onto proteins, an important posttranslational modification involved in transcription, DNA damage repair, and stem cell identity. Previous studies established the activation of PARP1 in response to DNA damage, but little is known about PARP1 regulation outside of DNA repair. We developed an assay for measuring PARP activity in cell lysates and found that the basal activity of PARP1 was highly variable across breast cancer cell lines, independent of DNA damage. Sucrose gradient fractionation demonstrated that PARP1 existed in at least three biochemically distinct states in both high- and low-activity lines. A discovered complex containing the NuA4 chromatin-remodeling complex and PARP1 was responsible for high basal PARP1 activity, and NuA4 subunits were required for this activity. These findings present a pathway for PARP1 activation and a direct link between PARP1 and chromatin remodeling outside of the DNA damage response

doi:10.1017/S0033583510000314 Printed in the United States of America Conformational dynamics of the molecular chaperoneHsp90

Abstract. The ubiquitous molecular chaperone Hsp90 makes up 1–2 % of cytosolic proteins and is required for viability in eukaryotes. Hsp90 affects the folding and activation of a wide variety of substrate proteins including many involved in signaling and regulatory processes. Some of these substrates are implicated in cancer and other diseases, making Hsp90 an attractive drug target. Structural analyses have shown that Hsp90 is a highly dynamic and flexible molecule that can adopt a wide variety of structurally distinct states. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis only shift the equilibria between a pre-existing set of conformational states. For bacterial, yeast and human Hsp90, there is a conserved three-state (apo–ATP–ADP) conformational cycle; however; the equilibria between states are species specific. In eukaryotes, cytosolic co-chaperones regulate the in vivo dynamic behavior of Hsp90 by shifting conformational equilibria and affecting the kinetics of structural changes and ATP hydrolysis. In this review, we discuss the structural and biochemical studies leading to ou

Available online at www.sciencedirect.com pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase

Available onlin