Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity

Abstract

Small heat shock proteins protect cells from stress presumably by acting as molecular chaperones. Here we report on the functional characterization of a developmentally regulated, heat-inducible member of the Xenopus small heat shock protein family, Hsp30C. An expression vector containing the open reading frame of the Hsp30C gene was expressed in Escherichia coli. These bacterial cells displayed greater thermoresistance than wild type or plasmid-containing cells. Purified recombinant protein, 30C, was recovered as multimeric complexes which inhibited heat-induced aggregation of either citrate synthase or luciferase as determined by light scattering assays. Additionally, 30C attenuated but did not reverse heat-induced inactivation of enzyme activity. In contrast to an N-terminal deletion mutant, removal of the last 25 amino acids from the C-terminal end of 30C severely impaired its chaperone activity. Furthermore, heat-treated concentrated solutions of the C-terminal mutant formed nonfunctional complexes and precipitated from solution. Immunoblot and gel filtration analysis indicated that 30C binds with and maintains the solubility of luciferase preventing it from forming heat-induced aggregates. Coimmunoprecipitation experiments suggested that the carboxyl region is necessary for 30C to interact with target proteins. These results clearly indicate a molecular chaperone role for Xenopus Hsp30C and provide evidence that its activity requires the carboxyl terminal region.

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…Comparable results were reported for Xenopus Hsp30C. Deletion of 25 amino acids from the carboxyl end resulted in reduced solubility and impaired chaperone function (82).…”

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

“…Figure 5 summarizes these data in graphic format and displays the different efficiencies of sHsps to act as molecular chaperones. Thus, the 4 main sHsps of D melanogaster can perform functions similar to sHsps of other organisms in in vitro chaperone assays (Horwitz 1992;Ehrnsperger et al 1997;Lee et al 1997;Haslbeck et al 1999;Fernando and Heikkila 2000;Lindner et al 2000;Abdulle et al 2002;Panensenko et al 2002;Van Montfort et al 2002).…”

“…The heat-induced aggregation assay was conducted essentially as described in Fernando and Heikkila (2000). CS (0.1 M; molecular weight [MW] 51,629; Sigma, Oakville, ON, Canada) or luciferase (0.1 M; MW 61,000; Promega, Madison, WI, USA) were heat-denatured at 42ЊC in the absence or presence of either His-Hsp22, Hsp22, Hsp23, Hsp26, Hsp27 (0.05 M, 0.1 M, or 0.5 M) or bovine albumin serum (BSA; 0.1 M; ICN Biochemicals, Costa Mesa, CA, USA) as a control for 90 minutes for CS or 30 minutes for luciferase.…”

“…In vitro, many sHsps can act as molecular chaperones, inhibiting stress-induced aggregation of different protein substrates (Ehrnsperger et al 1997;Lee et al 1997;Haslbeck et al 1999;Fernando and Heikkila 2000). In vivo, overexpression of sHsps has been reported to confer thermotolerance (Landry et al 1989;Rollet et al 1992;Kitagawa et al 2002), protection against tumor necrosis factor-␣-induced and caspase-dependent apoptosis (Mehlen et al 1996;Arrigo 1998;Samali et al 2001;Concannon et al 2003), and stabilization of cytoskeletal elements (Lavoie et al 1993;Wieske et al 2001;Mounier and Arrigo 2002).…”

Deamidation Affects Structural and Functional Properties of Human αA-Crystallin and Its Oligomerization with αB-Crystallin

“…Based on similarities with the structure of other Hsps, it is believed that the N-terminal region (residues 1-62 in ␣A-and 1-66 in ␣B-crystallin(s)) of ␣-crystallin forms an independently folded domain, whereas the C terminus (referred as the C-terminal extension; residues 143-173 in ␣A-and 147-175 in ␣B-crystallin) is flexible and unstructured (4). Previous reports show that the removal of N-terminal residues (56 residues) and C-terminal extensions (32-34 residues) of ␣A-and ␣B-crystallins lead to improper folding, reduced chaperone activity, and formation of trimers or tetramers (5)(6)(7)(8)(9). The ␣-crystallin domain is believed to be engaged in the subunitsubunit interactions, but the individual amino acids in subunit interactions and chaperone activity have not been fully identified.…”