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 γ-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 Aβ42 peptide (Ala-Thr) and the other to the non-pathogenic Aβ48 (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.
Highlights
Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure
We investigated the in vitro kinetics of a microbial intramembrane aspartyl protease 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)
Intramembrane proteolysis must be highly regulated in the cell [13], yet contrary to intuition and knowledge of soluble proteases [44, 45], data to date do not converge on recognition motifs for Intramembrane proteases (IPs) within their TM substrate(s)
Summary
To assess contributions of particular residues to catalysis (Table 1 and Fig. 1A; see below), we employed a continuous kinetics Förster resonance energy transfer (FRET) peptide assay in combination with a discontinuous gel-based assay, methodology we reported previously [26]. Consistent with our measurements (Table 1), one additional study of the G384A presenilin variant reported 2-fold reduced Vmax compared with WT enzyme [33] Rigidifying this region with the G219A substitution is detrimental to optimal orientation of Asp220 for catalysis, it is noteworthy that the reaction still proceeds. In terms of how substrate may gain entry into the active site, replacing the mIAP sequence AGL275 with PAL435 found in presenilin [23,24,25] reduces catalytic efficiency by ϳ80% compared with WT, indicating that PAL is not a favorable substitute (Fig. 1D and Table 1). S.D. Kinetic parameters for mIAP cleavage of C100FRET in DDM and bicelles and inhibition by (ZLL)2ketone
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