Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog

Swe Htet Naing, Sibel Kalyoncu, David M. Smalley, Hyojung Kim, Xingjian Tao, Josh B. George, Alex P. Jonke, Ryan C. Oliver, Volker S. Urban, Matthew P. Torres, Raquel L. Lieberman

Research output: Contribution to journalReview articlepeer-review

12 Scopus citations

Abstract

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of -secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated A42 peptide (Ala-Thr) and the other to the non-pathogenic A48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate- enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Original languageEnglish
Pages (from-to)4653-4663
Number of pages11
JournalJournal of Biological Chemistry
Volume293
Issue number13
DOIs
StatePublished - Mar 30 2018

Funding

1 Supported by a travel grant from the College of Sciences at Georgia Tech to collect data at ORNL. This work was funded by National Science Foundation Grant MCB 0845445 (to R. L. L.) and National Institutes of Health Grants R01GM112662 and R01GM118744 (to M. P. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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