Regulation of stress-induced intracellular sorting and chaperone function of Hsp27 (HspB1) in mammalian cells

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.

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

“…In protein refolding assays in vitro (Horwitz, 1992; Jakob et al ., 1993), aggregation prevention by the ATP‐independent small HSPs has been shown to occur independent of the ATP‐dependent HSP70 machinery, but for efficient refolding, substrate transfer to the HSP70 machine is required (Lee & Vierling, 2000; Mogk et al ., 2003). Also, in living cells, refolding assistance by HSPB1 was found to require a functional HSP70 machinery (Bryantsev et al ., 2007), and in this study, a similar scenario was found to be true for the best refolding stimulating D. melanogaster small HSP, CG14207. Strikingly, CG14207 was ineffective in preventing polyQ aggregation.…”

“…In vitro , the addition of the HSP70/HSP40 refolding machinery is required for the refolding reaction (Lee et al ., 1997). This has been reproduced in living cells using luciferase as a substrate (Nollen et al ., 2001; Bryantsev et al ., 2007). Here, we tailored the cellular luciferase refolding assay for Drosophila S2 cells and characterized which of the Drosophila small HSPs could enhance luciferase refolding upon overexpression.…”

“…To analyze whether D. melanogaster small HSPs, like bacterial, plant, and mammalian small HSPs (Lee & Vierling, 2000; Mogk et al ., 2003; Bryantsev et al ., 2007), also require HSP70 machines for refolding, we first tested which of the D. melanogaster HSP70s could promote refolding of heat‐denatured luciferase. Hereto, we cloned a selection of the D. melanogaster HSP70 family (Table S2, Supporting information) and analyzed their effect on luciferase refolding.…”

Characterization of hsp27 kinases activated by elevated aortic pressure in heart

“…Hsp27 is thought to play a role in protecting the myocardium during periods of stress and linkage analysis suggested hsp27 as a determinant of heart mass in spontaneously hypertensive rats [37]. The actin-capping, aggregation and, possibly molecular chaperone function, of hsp27 are regulated by phosphorylation (see [8,10]). The expression and/or phosphorylation of hsp27 in the heart is increased in response to chronic pressure overload [20], both physiological and pathological hypertrophy [13], heart failure [21,38,39], oxidative stress [24,25], ischemic injury [22], and haemorrhagic shock [23].…”

“…Hsp27 function appears to be regulated by posttranslational modifications, including phosphorylation at Ser-18, Ser-78, Ser-82, and Thr-143 [8,2]. Phosphorylation promotes the dissociation of hsp27 oligomers [9] and phospho-mimetic hsp27 mutants (i.e., S-15,78,82-D) show enhanced chaperone activity [10]. Furthermore, when phosphorylated, hsp27 dissociates from F-actin and may bind laterally to, and stabilize, actin filaments [7,11].…”