Activity-Regulated Cytoskeleton-Associated Protein (Arc/Arg3.1) is Transiently Expressed after Heat Shock Stress and Suppresses Heat Shock Factor 1.

Heat shock proteins are induced by activation of heat shock factor 1 (HSF1) in response to heat shock and protect against heat stress. However, the molecular mechanisms underlying the downstream signal of heat shock have not been fully elucidated. We found that similarly to canonical Hsps, Arc/Arg3.1 is also markedly induced by heat shock and by other cellular stress inducers, including diamide, sodium arsenite and H2O2 in various cells. We noted that heat stress-induced Arc/Arg3.1 protein is short lived, with a half-life of <30 min, and is readily degraded by the ubiquitin-proteasome system. Arc/Arg3.1 overexpression inhibited the up-regulation of heat shock-induced Hsp70 and Hsp27, suggesting that Arc/Arg3.1 is a negative regulator of heat shock response (HSR). Studying the effect of Arc/Arg3.1 on HSF1, a major transcription factor in HSR, we found that Arc/Arg3.1 binds to HSF1 and inhibits its binding to the heat shock element in gene promoters, resulting in reduced induction of Hsp27 and Hsp70 mRNAs, without affecting HSF1's phosphorylation-dependent activation, or nuclear localization. Arc/Arg3.1 overexpression decreased cell survival in response to heat shock. We conclude that Arc/Arg3.1 is transiently expressed after heat shock and negatively regulates HSF1 in the feedback loop of HSR.

Evidence for the dimerization-mediated catalysis of methionine sulfoxide reductase A from Clostridium oremlandii.

Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.

Hydrogen-deuterium exchange mass spectrometry for determining protein structural changes in drug discovery.

Protein structures are dynamically changed in response to post-translational modifications, ligand or chemical binding, or protein-protein interactions. Understanding the structural changes that occur in proteins in response to potential candidate drugs is important for predicting the modes of action of drugs and their functions and regulations. Recent advances in hydrogen/deuterium exchange mass spectrometry (HDX-MS) have the potential to offer a tool for obtaining such understanding similarly to other biophysical techniques, such as X-ray crystallography and high resolution NMR. We present here, a review of basic concept and methodology of HDX-MS, how it is being applied for identifying the sites and structural changes in proteins following their interactions with other proteins and small molecules, and the potential of this tool to help in drug discovery.