Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β (Aβ), peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulingrowth factor-II (IGF-II) and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor (EGF), respectively. Here, we used high accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and Aβ by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (aa 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and EGF families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their Nterminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.

Blood coagulation often accompanies bacterial infections and sepsis and is generally accepted as a consequence of immune responses. Though many bacterial species can directly activate individual coagulation factors, they have not been shown to directly initiate the coagulation cascade that precedes clot formation. Here we demonstrated, using microfluidics and surface patterning, that the spatial localization of bacteria substantially affects coagulation of human and mouse blood and plasma. Bacillus cereus and Bacillus anthracis, the anthrax-causing pathogen, directly initiated coagulation of blood in minutes when bacterial cells were clustered. Coagulation of human blood by B. anthracis required secreted zinc metalloprotease InhA1, which activated prothrombin and factor X directly (not via factor XII or tissue factor pathways). We refer to this mechanism as 'quorum acting' to distinguish it from quorum sensing-it does not require a change in gene expression, it can be rapid and it can be independent of bacterium-to-bacterium communication.This paper describes a physical and biochemical mechanism responsible for regulating the initiation of human blood coagulation by bacteria. In vivo, coagulation often accompanies bacterial infections of the blood and is believed to be a consequence of immune and inflammatory responses 1-5 . Immune and inflammatory responses cause upregulation of tissue factor on the timescale of hours and lead to increased coagulation 6,7 . One of the few drugs available to treat septic shock, activated protein C, is also an anticoagulant 8 . This coagulation is believed to prevent dissemination of bacteria through the blood 9,10 but also results in serious vascular damage due to blockage and injury of blood vessels 8 . Coagulation accompanying bacterial infections of the blood is particularly relevant for people infected with anthrax, which involves sepsis and disseminated intravascular coagulation caused by the pathogen Bacillus Correspondence should be addressed to R.F.I. (r-ismagilov@uchicago.edu).. AUTHOR CONTRIBUTIONS C.J.K., J.Q.B., M.M., Y.B., R.R.P., T.R.K. and F.S. performed experiments; C.J.K., J.Q.B., M.M., Y.B., R.R.P., T.R.K., F.S., S.H.L., W.-J.T. and R.F.I. designed experiments and analyzed data; C.J.K., W.-J.T. and R.F.I. wrote the paper; A.P.P. and P.S. provided reagents. NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2009 June 1. Published in final edited form as:Nat Chem Biol. 2008 December ; 4(12): 742-750. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript anthracis 4 . Here, we considered an alternative and complementary mechanism for the coagulation that accompanies infection: direct activation of the human coagulation cascade through activation of coagulation factors by bacteria.Many bacteria and bacterial components can directly activate individual human coagulation factors. However, direct initiation of the coagulation cascade and the formation of a propagating clot are not typically observed 11-17 ...

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