Abstract
Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (kcat = 2 s- 1) followed by a slow cleavage between residues 72 and 73 (kcat = 0.07 s- 1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG < 0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.
Original language | English (US) |
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Pages (from-to) | 454-466 |
Number of pages | 13 |
Journal | Journal of Molecular Biology |
Volume | 406 |
Issue number | 3 |
DOIs | |
State | Published - Feb 25 2011 |
Funding
This work was supported by National Institutes of Health grants GM 81539 to W.J.T., GM55694 to T.R.S., and GM36806 to R.D.G., by National Institutes of Health fellowship F32 GM 87093 to L.A.R., and by Arnold and Mabel Beckman Foundation to V.K. We are grateful to the staff of APS Structural Biology Center for help in data collection. Use of the APS was supported by the U.S. Department of Energy, Office of Basic Energy Sciences , under contract no. W-31-109-ENG-38 . Use of proteomics service facility was supported by Chicago Biomedical Consortium.
Keywords
- exosite
- insulin-degrading enzyme
- nestin-mediated cleavage regulation
- substrate flexibility
- ubiquitin turnover
ASJC Scopus subject areas
- Molecular Biology
- Biophysics
- Structural Biology
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Crystal structure of human insulin-degrading enzyme in complex with ubiquitin
Ralat, L. A. (Contributor), Kalas, V. (Contributor), Zheng, Z. (Contributor), Goldman, R. D. (Contributor), Sosnick, T. R. (Contributor) & Tang, W.-J. (Contributor), Protein Data Bank (PDB), Sep 8 2010
DOI: 10.2210/pdb3OFI/pdb, https://www.wwpdb.org/pdb?id=pdb_00003ofi
Dataset