STUB1

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.

Abstract: Molecular chaperones, ubiquitin ligases and proteasome impairment have been implicated in several neurodegenerative diseases, including Alzheimer's and Parkinson's disease, which are characterized by accumulation of abnormal protein aggregates (e.g. tau and alpha-synuclein respectively). Here we report that CHIP, an ubiquitin ligase that interacts directly with Hsp70/90, induces ubiquitination of the microtubule associated protein, tau. CHIP also increases tau aggregation. Consistent with this observation, diverse of tau lesions in human postmortem tissue were found to be immunopositive for CHIP. Conversely, induction of Hsp70 through treatment with either geldanamycin or heat shock factor 1 leads to a decrease in tau steady-state levels and a selective reduction in detergent insoluble tau. Furthermore, 30-month-old mice overexpressing inducible Hsp70 show a significant reduction in tau levels. Together these data demonstrate that the Hsp70/CHIP chaperone system plays an important role in the regulation of tau turnover and the selective elimination of abnormal tau species. Hsp70/CHIP may therefore play an important role in the pathogenesis of tauopathies and also represents a potential therapeutic target.

Abstract: The ubiquitin-proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C-terminus of Hsc70-interacting protein (CHIP) with a U-box domain is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U-box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C-terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self-ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as 'a quality-control E3' that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.