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Molecular determinants of substrate specificity in human insulin-degrading enzyme Lazaros Stefanidis, Nicholas D. Fusco, Samantha E. Cooper, Jillian Smith-Carpenter, and Benjamin J. Alper Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00474 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018
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Biochemistry
Molecular Determinants of Substrate Specificity in Human Insulin-Degrading Enzyme Lazaros Stefanidis1, Nicholas D. Fusco1, Samantha E. Cooper2, Jillian E. Smith-Carpenter2 and Benjamin J. Alper1* From the 1Department of Chemistry, Sacred Heart University, Fairfield CT, USA 06825; 2Department of Chemistry and Biochemistry, Fairfield University, Fairfield CT 06824 Running title: Functional analysis of the IDE substrate binding interface *To whom correspondence should be addressed: Benjamin J. Alper: Department of Chemistry, Sacred Heart University, Fairfield CT 06825;
[email protected]; Tel. (203) 396-8105, Fax. (203) 371-7888. Keywords: insulin-degrading enzyme, M16A metalloendopeptidase, insulin, amyloid peptide
ABSTRACT Insulin-degrading enzyme (IDE) is a 110 kDa chambered zinc metalloendopeptidase that degrades insulin, amyloid beta, and other intermediate-sized aggregation prone peptides that adopt β-structures. Structural studies of IDE in complex with multiple physiological substrates have suggested a role for hydrophobic and aromatic residues of the IDE active site in substrate binding and catalysis. Here, we examine functional requirements for conserved hydrophobic and aromatic IDE active site residues that are positioned within 4.5 Angstroms of IDE bound insulin B chain and amyloid beta peptides in the reported crystal structures for the respective enzyme-substrate complexes. Charge, size, hydrophobicity, aromaticity, and other functional group requirements for substrate binding IDE active site residues were examined through mutational analysis of the recombinant human enzyme and enzyme kinetic studies conducted using native and fluorogenic derivatives of human insulin and amyloid beta peptides. A functional requirement for IDE active site residues F115, A140, F141, Y150, W199, F202, F820, and Y831 was established, and specific contributions of residue charge, size and hydrophobicity in substrate binding, specificity, and proteolysis were demonstrated. IDE mutant alleles that exhibited enhanced or diminished proteolytic activity towards insulin or amyloid beta peptides and derivative substrates were identified.
INTRODUCTION Human insulin-degrading enzyme (IDE; EC 3.4.24.56, GenBank reference sequence BC096336) is a 110 kDa chambered zinc metalloendopeptidase of the M16A subfamily. The enzyme is notable for its role in the proteolysis of several physiologically significant peptides that adopt β-structures, including insulin (1, 2), glucagon (3), amylin (4) and amyloid beta (Aβ) peptides (5). 1
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Functional analysis of the IDE substrate binding interface Multiple studies point to roles for IDE in metabolic signaling and disease. Consistent with a role for IDE within the insulin signaling pathway, human IDE polymorphisms have been associated with type 2 diabetes (6, 7), and in IDE deficient mouse models, loss of IDE corresponds with elevated serum insulin and glucose levels, and intolerance to glucose challenge after fasting (8, 9). Moreover, in accordance with the amyloid hypothesis, which asserts that variations in neuronal Aβ production and catabolism contribute to the development of Alzheimer’s disease (10, 11, 12), IDE is thought to play a neuroprotective role in the clearance of free Aβ peptides (8, 13, 14, 15). Proteolytic clearance of Aβ peptides by IDE is hypothesized to limit peptide accumulation, plaque formation and neurotoxicity (16, 17, 18, 19). Consistent with this hypothesis, transgenic mice that overexpress amyloid precursor protein exhibit cognitive deficits, whereas neuronal levels of Aβ and premature death rates are significantly reduced in transgenic mice where IDE is overexpressed in parallel (20), and primary neurons cultured from IDE knockout mice show >90% reduction in the rate of Aβ degradation (8). IDE is a member of the M16A subfamily of zinc metalloendopeptidases. M16A peptidases are broadly conserved, with homologs expressed throughout the prokaryotic and eukaryotic kingdoms (21, 22). M16A enzymes hydrolyze intermediate-sized (
