No abstract

In vitro, small Hsps (heat-shock proteins) have been shown to have chaperone function capable of keeping unfolded proteins in a form competent for Hsp70-dependent refolding. However, this has never been confirmed in living mammalian cells. In the present study, we show that Hsp27 (HspB1) translocates into the nucleus upon heat shock, where it forms granules that co-localize with IGCs (interchromatin granule clusters). Although heat-induced changes in the oligomerization status of Hsp27 correlate with its phosphorylation and nuclear translocation, Hsp27 phosphorylation alone is not sufficient for effective nuclear translocation of HspB1. Using firefly luciferase as a heat-sensitive reporter protein, we demonstrate that HspB1 expression in HspB1-deficient fibroblasts enhances protein refolding after heat shock. The positive effect of HspB1 on refolding is completely diminished by overexpression of Bag-1 (Bcl-2-associated athanogene), the negative regulator of Hsp70, consistent with the idea of HspB1 being the substrate holder for Hsp70. Although HspB1 and luciferase both accumulate in nuclear granules after heat shock, our results suggest that this is not related to the refolding activity of HspB1. Rather, granular accumulation may reflect a situation of failed refolding where the substrate is stored for subsequent degradation. Consistently, we found 20S proteasomes concentrated in nuclear granules of HspB1 after heat shock. We conclude that HspB1 contributes to an increased chaperone capacity of cells by binding unfolded proteins that are hereby kept competent for refolding by Hsp70 or that are sorted to nuclear granules if such refolding fails.

. Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion. Am J Physiol Cell Physiol 283: C521-C534, 2002. First published March 27, 2002 10.1152/ ajpcell.00503.2001.-Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress. chaperone; protein aggregation; metabolic stress; ischemic tolerance AT THE CELLULAR LEVEL, failing energy metabolism and ATP depletion are the earliest cell-damaging factors of ischemic insults. In vivo, severe depletion of ATP is a proteotoxic stress that leads to dysfunction, destabilization, and aggregation of many cellular proteins, including enzymes, ion pumps, and constituents of cytoskeletal and contractile structures (1,9,17,27). Sustained lack of ATP is obviously lethal for the cell.On the contrary, a transient (reversible) drop in cellular ATP can confer tolerance to the next energy-depriving exposure, with heat shock proteins (HSPs) being involved in such an adaptive response (reviewed in Ref. 17). The HSP-involving cellular response appears to contribute to delayed ischemic tolerance (the second window of protection) found after heat or ischemic preconditioning in the myocardium. Like high temperature, cellular ATP depletion activates the heat shock transcription factor 1 (HSF1) that afterward induces HSP expression in the recovering cells (4, 36). Most HSPs are molecular chaperones stabilizing protein molecules under heat shock conditions in vitro and in vivo, and the same chaperone activity may protect HSP-enriched cells in the case of other proteotoxic stresses, e.g....

Overexpression of heat shock protein (Hsp) 70 and Hsp27 in vivo was proclaimed as a potential tool in therapy of ischemia-reperfusion injury. However, it was so far not known whether these Hsps can beneficially act when increased in cells just at the stage of postischemic reperfusion. This issue was examined in a model of ischemia-reperfusion stress when cultures of endothelial cells (EC) from human umbilical vein were infected with virus-based vectors expressing Hsp70 or Hsp27, or Hsp56, or green fluorescent protein (GFP) and exposed to 20 hours of hypoxia followed by reoxygenation. The infection was performed either 10 hours before hypoxia or immediately after hypoxia, or at different time points of reoxygenation. Only low cell death was detected during hypoxia, but later, up to 40% of the treated cells died via caspase-dependent apoptosis between 6 and 12 hours of reoxygenation. The percentage of apoptotic cells was 1.6- to 3-fold greater in Hsp56- and GFP-infected EC than in Hsp70- or Hsp27-infected EC. The last 2 groups exhibited a lesser extent of procaspase-9 and procaspase-3 activation within 6-9 hours of reoxygenation. The cytoprotective effects of overexpressed Hsp70 and Hsp27 were observed not only in the case of infection before hypoxia but also when EC were infected at the start of reoxygenation or 1-2 hours later. An increase in the Hsp70 and Hsp27 levels in infected EC correlated well with their resistance to apoptosis under reoxygenation. These findings suggest that overexpression of Hsp70 or Hsp27, if it occurs in the involved cells at the early stage of postischemic reperfusion, can still be cytoprotective.

Previously we have found that stationary Ehrlich ascites carcinoma (EAC) cells in vivo accumulated heat shock proteins (HSPs) and became resistant to necrotic death induced by prolonged energy deprivation of hyperthermia. Here we report that apoptotic death induced by nutrient starvation, transient ATP depletion, heat shock and a microtubule-disrupting drug, vinblastine, was also suppressed in stationary EAC cells comparing with exponential cells. When exponential (sensitive) cells were subjected to short-term heating with recovery to accumulate inducible form of HSP70, they also became resistant to all of the employed apoptosis-inducing exposures, and an inhibitor of cytosolic protein synthesis, cycloheximide, prevented acquisition of the resistance. It is suggested that in vivo accumulation of HSPs in stationary tumor cells can be endogenous protective device against apoptotic death induced by starvation or some anticancer treatments.