Riluzole Increases the Amount of Latent HSF1 for an Amplified Heat Shock Response and Cytoprotection

BACKGROUND: Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases. METHODOLOGY/PRINCIPAL FINDINGS: We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNA(hsf1) by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [(35)S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect. CONCLUSION/SIGNIFICANCE: We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention

Activator-induced dynamic disorder and molecular memory in human two-pore domain hTREK1 K+ channel

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. Ion channel flux activity is comparable to the catalytic activity of enzyme molecules. Saturating concentrations of substrate induce “dynamic disorder” in the kinetic rate processes of single-enzyme molecules and consequently, develop correlative “memory” of the previous history of activities. Similarly, binding of ions as substrate alone or in presence of agonists affects the catalytic turnover of single-ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in the single human-TREK1-channel due to binding of substrate/agonist using the excised inside–out patch-clamp technique. Our results suggest that the single-hTREK1-channel behaves as a typical Michaelis–Menten enzyme molecule with a high-affinity binding site for K+ ion as substrate. But, in contrast to enzyme, dynamic disorder in single-hTREK1-channel was not induced by substrate K+ binding, but required allosteric modification of the channel molecule by the agonist, trichloroethanol. In addition, interaction of trichloroethanol with hTREK1 induced strong correlation in the waiting time and flux intensity, exemplified by distinct mode-switching between high and low flux activities. This suggested the induction of molecular memory in the channel molecule by the agonist, which persisted for several decades in time. Our mathematical modeling studies identified the kinetic rate processes associated with dynamic disorder. It further revealed the presence of multiple populations of distinct conformations that contributed to the “heterogeneity” and consequently, to the molecular memory phenomenon that we observed