title: Protective role of Cys-178 against the inactivation and oligomerization of human insulin-degrading enzyme by oxidation and nitrosylation.
abstract: Insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, hydrolyzes several physiologically relevant peptides, including insulin and amyloid-beta (Abeta). Human IDE has 13 cysteines and is inhibited by hydrogen peroxide and S-nitrosoglutathione (GSNO), donors of reactive oxygen and nitrogen species, respectively. Here, we report that the oxidative burst of BV-2 microglial cells leads to oxidation or nitrosylation of secreted IDE, leading to the reduced activity. Hydrogen peroxide and GSNO treatment of IDE reduces the V(max) for Abeta degradation, increases IDE oligomerization, and decreases IDE thermostability. Additionally, this inhibitory response of IDE is substrate-dependent, biphasic for Abeta degradation but monophasic for a shorter bradykinin-mimetic substrate. Our mutational analysis of IDE and peptide mass fingerprinting of GSNO-treated IDE using Fourier transform-ion cyclotron resonance mass spectrometer reveal a surprising interplay of Cys-178 with Cys-110 and Cys-819 for catalytic activity and with Cys-789 and Cys-966 for oligomerization. Cys-110 is near the zinc-binding catalytic center and is normally buried. The oxidation and nitrosylation of Cys-819 allow Cys-110 to be oxidized or nitrosylated, leading to complete inactivation of IDE. Cys-789 is spatially adjacent to Cys-966, and their nitrosylation and oxidation together trigger the oligomerization and inhibition of IDE. Interestingly, the Cys-178 modification buffers the inhibition caused by Cys-819 modification and prevents the oxidation or nitrosylation of Cys-110. The Cys-178 modification can also prevent the oligomerization-mediated inhibition. Thus, IDE can be intricately regulated by reactive oxygen or nitrogen species. The structure of IDE reveals the molecular basis for the long distance interactions of these cysteines and how they regulate IDE function.
authors.affiliation: Ben May Department for Cancer Research, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Ralat
authors.forename: Min
authors.initials: M
authors.lastname: Ren
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Schilling
authors.forename: Wei-Jen
authors.initials: WJ
authors.lastname: Tang
mesh_terms: D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007339:Insulysin / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D017628:Microglia / Q000378:metabolism; D008954:Models, Biological; D009584:Nitrogen / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D016897:Respiratory Burst; D026422:S-Nitrosoglutathione / Q000737:chemistry
chemical_list: D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D026422:S-Nitrosoglutathione; D006861:Hydrogen Peroxide; D007339:Insulysin; D003545:Cysteine; D009584:Nitrogen; D010100:Oxygen