Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

TLDR

The 70‑kDa heat shock proteins Hsc70 and Hsp70 are regulated by cofactors Hsp40, BAG‑1, Hip, and Hop, all of which interact through tetratricopeptide repeat domains. The study aimed to identify additional tetratricopeptide repeat‑containing proteins and discovered a novel 35‑kDa cytoplasmic protein, carboxyl‑terminus of Hsc70‑interacting protein (CHIP). CHIP was identified by yeast two‑hybrid screening, binds the C‑terminal 540–650 residues of Hsc70, interacts reciprocally with CHIP’s N‑terminal 1–197 residues, and inhibits Hsp40‑stimulated ATPase activity of Hsc70 and Hsp70. CHIP is highly expressed in adult striated muscle, binds Hsc70/Hsp70 in vitro and in vivo, and its interaction decreases ATPase activity and chaperone efficiency, indicating negative regulation of the Hsc70‑Hsp70 substrate‑binding cycle.

Abstract

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.