Differences in the chaperone-like activities of the four main small heat shock proteins of Drosophila melanogaster

Abstract

The Drosophila melanogaster family of small heat shock proteins (sHsps) is composed of 4 main members (Hsp22, Hsp23, Hsp26, and Hsp27) that display distinct intracellular localization and specific developmental patterns of expression in the absence of stress. In an attempt to determine their function, we have examined whether these 4 proteins have chaperone-like activity using various chaperone assays. Heat-induced aggregation of citrate synthase was decreased from 100 to 17 arbitrary units in the presence of Hsp22 and Hsp27 at a 1:1 molar ratio of sHsp to citrate synthase. A 5 M excess of Hsp23 and Hsp26 was required to obtain the same efficiency with either citrate synthase or luciferase as substrate. In an in vitro refolding assay with reticulocyte lysate, more than 50% of luciferase activity was recovered when heat denaturation was performed in the presence of Hsp22, 40% with Hsp27, and 30% with Hsp23 or Hsp26. These differences in luciferase reactivation efficiency seemed related to the ability of sHsps to bind their substrate at 42ЊC, as revealed by sedimentation analysis of sHsp and luciferase on sucrose gradients. Therefore, the 4 main sHsps of Drosophila share the ability to prevent heat-induced protein aggregation and are able to maintain proteins in a refoldable state, although with different efficiencies. The functional reasons for their distinctive cell-specific pattern of expression could reflect the existence of defined substrates for each sHsp within the different intracellular compartments.

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

“…The small HSPs tested so far (HSP22, HSP23, HSP26, HSP27) share the capacity to facilitate refolding of stress‐denatured substrates in vitro (Morrow et al ., 2006), supporting the hypothesis that maintenance of global protein homeostasis is essential for longevity. To elucidate which Drosophila small HSPs might be the most potent in preventing protein misfolding or the toxic aggregation of protein damage upon aging, we cloned all members of the Drosophila small HSP family (excluding the mitochondrial HSP22) and compared their ability to assist in refolding of stress‐denatured substrates and/or in preventing aggregation of disease‐associated misfolded proteins in living cells.…”

“…2, bottom) although expression of HSP23 and HSP26 was somewhat higher and expression of CG4461 was somewhat lower than that of the other seven sHSPs that showed similar expression levels. Consistent with in vitro data (Morrow et al ., 2006), overexpression of the classical small HSPs (HSP23, HSP26, and HSP27) increased luciferase refolding (Fig. 2, top).…”

Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders

“…HSP70s/HSPAs), the HSPB-bound clients can either be refolded or degraded; the mechanisms for either refolding or degradation is not fully understood, but may depend both on the state of the client and on the specific HSPB member that is bound to it (see later). The chaperone activity of small HSPs has been discovered and explored mainly in cell-free experiments with purified proteins [58][59][60] and it accounts for, for example, the role that HSPB4 plays in maintaining eye transparency [26]. Whether this chaperone function is also underlying other cellular functions of HSPB members is less clear.…”

The Hsp27 gene is not required for Drosophila development but its activity is associated with starvation resistance

“…A number of sHSPs form large oligomers in cells and the oligomers bind to unfolded proteins (Van Montfort et al 2001; Giese and Vierling 2002). Investigations into the mechanisms of unfolding and refolding proteins suggest that the primary function of sHSPs is to protect denatured proteins from aggregation by forming complexes with malfolded polypeptides (Jakob and Buchner 1994;Ehrnsperger et al 2000;Morrow et al 2006). sHSPs are implicated in a variety of cellular activities, including thermotolerance, resistance to apoptosis, and eye lens transparency (Landry et al 1989;Arrigo 1998;Liang and MacRae 1999;Andley et al 2002).…”