Molecular basis for the thiol sensitivity of insulin-degrading enzyme.
Insulin-degrading enzyme (IDE) is a ubiquitous zinc-metalloprotease that hydrolyzes several pathophysiologically relevant peptides, including insulin and the amyloid beta-protein (Abeta). IDE is inhibited irreversibly by compounds that covalently modify cysteine residues, a mechanism that could be operative in the etiology of type 2 diabetes mellitus (DM2) or Alzheimer's disease (AD). However, despite prior investigation, the molecular basis underlying the sensitivity of IDE to thiol-alkylating agents has not been elucidated. To address this topic, we conducted a comprehensive mutational analysis of the 13 cysteine residues within IDE. Our analysis implicates C178, C812, and C819 as the principal residues conferring thiol sensitivity. The involvement of C812 and C819, residues quite distant from the catalytic zinc atom, provides functional evidence that the active site of IDE comprises two separate domains that are operational only in close apposition. Structural analysis and other evidence predict that alkylation of C812 and C819 disrupts substrate binding, whereas alkylation of C178 interferes with the apposition of active-site domains and subtly repositions zinc-binding residues. Unexpectedly, alkylation of C590 was found to activate hydrolysis of Abeta significantly, while having no effect on insulin, demonstrating that chemical modulation of IDE can be both bidirectional and highly substrate selective. Our findings resolve a long-standing riddle about the basic enzymology of IDE with important implications for the etiology of DM2 and AD. Moreover, this work uncovers key details about the mechanistic basis of the unusual substrate selectivity of IDE that may aid the development of pharmacological agents or IDE mutants with therapeutic value.
Neant-Fery
Marie
M
Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Garcia-Ordoñez
Rubén D
RD
Logan
Todd P
TP
Selkoe
Dennis J
DJ
Li
Lilin
L
Reinstatler
Lael
L
Leissring
Malcolm A
MA
D000477:Alkylating Agents / Q000494:pharmacology; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D007339:Insulysin / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D000477:Alkylating Agents; D013438:Sulfhydryl Compounds; D007339:Insulysin; D003545:Cysteine