The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase

Bolduc, David M.; Montagna, Daniel R.; Seghers, Matthew; Wolfe, Michael S.; Selkoe, Dennis J.

doi:10.7554/elife.17578

Cited by 147 publications

(228 citation statements)

References 37 publications

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“…As shown in Figure 2, the compound with a p3’ substituent inhibited γ-secretase activity with an IC 50 of roughly 1 µM under these conditions, while the compound lacking a p3’ substituent did not inhibit γ-secretase activity at any concentration tested, confirming the data obtained in cells suggesting the presence of an S3’ pocket on γ-secretase. Once the substrate binds to the γ-secretase active site for proteolysis, these three hydrophobic pockets can accommodate three residues downstream of the scissile amide bond, explaining why carboxypeptidase trimming of long Aβ intermediates occurs in intervals of 3 amino acids 47 . The three S’ pocket model could also potentially explain why ε cleavage primarily occurs exactly three residues within the CTFβ TMD to generate Aβ49 (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

et al. 2016

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The amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1’, S2’, and S3’ pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

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Section: Resultsmentioning

confidence: 99%

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

et al. 2016

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“…While helix-breaking residues within the TM segment were once thought to be important for cleavage (18,20,47), examples to the contrary appear in the literature (10,11). Confounding the puzzle for IAPs is that IAPs appear to cleave at multiple cut sites, and/or trim TM helices to smaller segments that can eventually be released from the membrane (27,(48)(49)(50). Systematic in vitro studies using purified model enzymes should help clarify the physicochemical preferences of IAPs toward their substrates.…”

Section: Discussionmentioning

confidence: 99%

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

Naing

Kalyoncu

Smalley³

et al. 2018

Journal of Biological Chemistry

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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-

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“…Subsequently, γ‐secretase breaks down the remaining part of C‐terminal fragment of APP through a stepwise trimming process generating an array of Aβ peptides of different lengths (Morishima‐Kawashima ; Bolduc et al . ). The two major peptides are Aβ40 and Aβ42.…”

Section: Hypoxia Activates γ‐Secretasementioning

confidence: 97%

Hypoxia/ischemia activate processing of Amyloid Precursor Protein: impact of vascular dysfunction in the pathogenesis of Alzheimer's disease

Salminen

Kauppinen

Kaarniranta

2017

Journal of Neurochemistry

170

124

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Alzheimer's disease (AD) is associated with deficiencies in cerebrovascular functions, e.g. reduced cerebral blood flow and capillary amyloid angiopathy, both of which are evident during the early phase of AD, thus local hypoxia/ischemia could augment the pathogenesis of AD. There is abundant literature revealing that exposures to hypoxia/ischemia increase the amyloidogenic processing of amyloid-b precursor protein (APP) leading to the accumulation of amyloid-b peptides in brain. This hypoxia-induced response has been attributed to a significant increase in the activities of b-and csecretases, whereas a-secretase activity decreases in hypoxia. Recent studies have indicated that hypoxia-inducible factor-1a (HIF-1a) stimulates the transcription of the bsecretase 1 (BACE1) gene through the hypoxia-response element in the BACE1 promoter. Moreover, HIF-1a protein can directly interact with the c-secretase complex and increase its activity in a non-transcriptional manner. Hypoxia/ischemia also trigger endoplasmic reticulum stress and impair autophagy in brain, which consequently can stimulate the expression of presenilin 1 (PS1) and activate c-secretase. Subsequently, PS1 protein can stabilize HIF-1a protein and in addition, APP intracellular domain peptide is able to induce the expression of HIF-1a. The activation of b-and c-secretases is an evolutionarily conserved hypoxia response, e.g. it is also present in zebrafish. Given that b-and c-secretases have many substrates in addition to APP, one could postulate that AD pathology is a byproduct of the rescue process mediated by these two aspartyl proteinases under hypoxic/ischemic conditions. We will review the recent evidence indicating that vascular dysfunctions can provoke AD pathology by activating b-and c-secretases.

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The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase

Cited by 147 publications

References 37 publications

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

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

Hypoxia/ischemia activate processing of Amyloid Precursor Protein: impact of vascular dysfunction in the pathogenesis of Alzheimer's disease

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