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

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 its hydroxyl substituent, in 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 the nonpathogenic 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 indicate 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. INTRODUCTIONIntramembrane proteases (IPs) cleave within a transmembrane (TM) helix of membranebound substrates to release cytoplasmic or extracellular proteins/peptides, which in turn translocate to different regions of the cell where they elicit their corresponding biological response related to, for example, cell differentiation, development, and metabolism (1). Despite their broad biomedical reach, basic questions surrounding the structure of the active enzyme, how substrates are presented, and how hydrolysis chemistry occurs in an active site sequestered within the hydrophobic lipid membrane, remain active areas of research.The least biochemically understood IP is the intramembrane aspartyl protease (IAP) enzyme class, one of just three bona fide IP types that hydrolyze substrates within the hydrophobic lipid environment by using different catalytic nucleophiles (2). IAPs employ membrane-

Insights into the catalytic properties of the mitochondrial rhomboid protease PARL

“…To further evaluate the cleavage of other known substrates of PARL, we generated internally quenched (IQ) peptide substrates (Arutyunova et al, 2018;Lapek et al, 2019;Naing et al, 2018;Ticha et al, 2017a) based on the amino acids flanking the PARL cleavage sites of PINK1 (Deas et al, 2011), PGAM5 (Sekine et al, 2012), and Smac (Saita et al, 2017). Kinetic analysis using both full-length and -truncated PARL with IQ-PINK1 [99][100][101][102][103][104][105][106][107][108] , IQ-PGAM5 [20][21][22][23][24][25][26][27][28][29] , and IQ-Smac 51-60 peptide substrates was first performed in detergent, which revealed similar Michaelis-Menten kinetics for all peptides (Fig 1F).…”

The metastable XBP1u transmembrane domain defines determinants for intramembrane proteolysis by signal peptide peptidase

“…7A). Likewise, recent kinetic measurements using the purified archaeal PSH showed a preference for cleavage at a threonine at the scissile peptide bond (Naing et al, 2018). Although the mechanism of substrate selection may be different, in an intriguing parallel also rhomboid proteases combine recognition of a defined cleavage site motif (Strisovsky et al, 2009) with sampling TM domain dynamics (Akiyama and Maegawa, 2007;Moin and Urban, 2012;Strisovsky, 2016;Urban and Freeman, 2003).…”

Taking a position on intramembrane proteolysis

“…The work from Naing et al (3) provides several new insights into IAPs and highlights similarities and differences to other intramembrane proteases. For example, positioning of the substrate recognition motif was less important for rhomboid proteases: Cleavage could even be shifted to positions outside of the transmembrane region by altering the location of the recognition motif (5).…”

“…Most studies of IAPs have focused on presenilin, but the ␥-secretase complex is a tetrameric heterologous oligomer, adding further challenges to a difficult system! Naing et al (3) instead focus their attention on the microbial IAP (mIAP) from Methanoculleus marisnigri, a presenilin homolog that acts with no known protein co-factors, providing a simplistic view of a still complicated system. The authors employ two different substrates: a presenilin substrate derived from amyloid precursor protein (APP) and a renin peptide, which is a soluble model substrate that is fortuitously cleaved by mIAP.…”

“…The intramembrane aspartyl protease (IAP) 2 presenilin, the enzymatic component of the gamma secretase complex known to cleave amyloid precursor protein, has 90 different known substrates that share no homology or consensus recognition motif (2), suggesting other factors are at play in determining substrate specificity and cleavage. A new study by Naing et al (3) provides insights into these possible factors in a detailed exploration of catalytic parameters, discovering both chemistry and positioning of substrate matters for hydrolysis.…”