期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 43, 页码 31069-31079出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.479345
关键词
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资金
- National Institutes of Health [DK51818, GM53457]
- American Cystic Fibrosis Foundation
- Manpei Suzuki Diabetes Foundation
- Translational Systems Biology and Medicine Initiative (TSBMI),Ministry of Education, Science, Sports and Culture (MEXT), Japan
- Grants-in-Aid for Scientific Research [22229009, 24650442, 24116507, 24710225] Funding Source: KAKEN
The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligaseactivity. In a cell-free reconstituted ER- associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I) EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.
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