γ-Secretase Substrate Concentration Modulates the Aβ42/Aβ40 Ratio

Presenilin (PS)/␥-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid ␤ (A␤)peptides in which A␤ species of different lengths are generated through multiple cleavages at the ␥-, -, and ⑀-sites. An increased A␤42/A␤40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased A␤42/A␤40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of ␥-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS ؊/؊ ) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo A␤ generation, suggesting that mutation impairs the total catalytic rate of ␥-secretase. Analysis of each A␤ species revealed that the FAD mutation specifically reduced A␤40 levels more drastically than A␤42 levels, leading to an increased A␤42/A␤40 ratio. By contrast, the FAD mutation increased the generation of longer A␤ species such as A␤43, A␤45, and >A␤46. These results were confirmed by analyses of ␥-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo A␤ generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased A␤42/A␤40 ratio observed in cases of FAD mutations is related to the differential inhibition of ␥-site cleavage reactions, in which the reaction producing A␤40 is subject to more inhibition than that producing A␤42. Our results also provide novel insight into how enhancing the generation of longer A␤s may contribute to Alzheimer disease onset.Amyloid ␤ (A␤) 2 is a hydrophobic peptide that pathologically deposits in the brains of Alzheimer disease (AD) patients. The significant neurotoxicity of oligomeric and/or fibrillar A␤ aggregates indicates that accumulation of A␤ is a central pathogenic event in AD (1). Sequential cleavage of ␤-amyloid precursor protein (APP) by ␤-and ␥-secretases releases A␤ into the luminal/extracellular space. ␤-secretase is a membranebound aspartic protease (identified as BACE) that cleaves the extracellular region of APP to produce membrane-spanning APP C-terminal fragment ␤ (termed CTF␤ or C99) and a N-terminal secreted form of APP␤. The intramembranous region of CTF␤ is cleaved next by ␥-secretase to produce A␤ and APP intracellular domain, and because of the loose site specificity of ␥-secretase-mediated proteolysis, various C-terminal truncated A␤ species, including two major species, A␤40 and A␤42, are also generated (1). A␤40 is the most predominant species of secreted A␤. A␤42 is hypothesized to be the trigger species for AD-related amyloid pathophysiology, because it has much faster aggregation potential than A␤40. Indeed, histochemical and biochemical studies have revealed that A␤42 primarily deposits within the brains of AD patients and several animal models (2-4).

Section: Enzymatic Characteristics Of Wild-type Ps1/␥-secretase In Psmentioning

confidence: 86%

Enzymatic Characteristics of I213T Mutant Presenilin-1/γ-Secretase in Cell Models and Knock-in Mouse Brains

Shimojo

Sahara

Mizoroki

et al. 2008

“…Protein concentration was determined with the DC Protein Assay kit (Bio-Rad) according to the manufacturer's instructions. ␥-Secretase activity was measured by electrochemiluminescence as described previously (18,33). CHAPSOsolubilized membrane was incubated in Buffer B (50 mM PIPES, pH 7.0, 150 mM KCl, 5 mM CaCl 2 , 5 mM MgCl 2 , and protease inhibitors) with 0.25% (v/v) CHAPSO, 1 M substrate, and 0.1% bovine serum albumin (v/v) in the presence or absence of ␥-secretase inhibitors for 2.5 h 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Total membrane fractions isolated from the Pen2 and APP cell lines were incubated with the APP transmembrane substrate, and A␤40 and A␤42 cleavage products were measured (33). Production of A␤40 was greatly reduced in the Pen2 cell line, but there was no effect on the production of A␤42 (Fig.…”

Section: Pen2 Overexpression Causes An Increase In the A␤42:a␤40mentioning

confidence: 99%

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 γ-Secretase Complexes

Placanica

Tarassishin

Yang

et al. 2009

Self Cite

␥-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic A␤42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of ␥-secretase, invariably increase the A␤42:A␤40 ratio. However, the mechanism by which these mutations affect ␥-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the A␤42:A␤40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active ␥-secretase complexes. Furthermore we reveal that the equilibrium of PS1-and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased A␤42:A␤40 ratios. These data suggest that perturbations to ␥-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased A␤42:A␤40 ratio.

“…The assays were performed as previously described (17,19,20) using electrochemiluminescence (ECL) technology. The amount of product was determined using synthetic peptide or recombinant standards.…”

Section: Methodsmentioning

confidence: 99%

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Tian

Crump

2010

Self Cite

Processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases generates pathogenic ␤-amyloid (A␤) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by ␣-secretases precludes A␤ formation. Little is known about the role of ␣-secretase cleavage in ␥-secretase regulation. Here, we show that ␣-secretase-cleaved APP C-terminal product (␣CTF) functions as an inhibitor of ␥-secretase. We demonstrate that the substrate inhibitory domain (ASID) within ␣CTF, which is bisected by the ␣-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns ␣CTF into a better substrate for ␥-secretase. Moreover, ␣-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of ␥-secretase activity by ␣CTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for ␣-secretase in generating an endogenous ␥-secretase inhibitor that down-regulates the production of A␤. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.The amyloid precursor protein (APP) 2 is sequentially cleaved by ␤-and ␥-secretases to generate A␤ peptides, which are widely considered to play a causative role in the pathogenesis of Alzheimer disease (AD) (1). This pathway also generates soluble APP␤ (sAPP␤) and APP intracellular domain. Alternatively, APP can be processed by ␣-secretase that cuts within A␤ peptides, which precludes the formation of toxic peptides. Similarly, the ␣/␥ pathway leads to formation of soluble APP␣ (sAPP␣), the P3 peptide, and the APP intracellular domain. It appears that ␥-secretase cleavage of substrates first requires another protease that removes the majority of the extracellular domain of the substrate. The sequential cleavage of APP is characteristic of regulated intramembrane proteolysis (RIP) (2). RIP generally requires two proteolytic steps, whereby the second intramembrane cleavage is dependent on the first cleavage, such as in Notch and sterol regulatory element binding proteins signaling. RIP has been found to govern sterol regulation, cell fate determination, unfolded protein responses, growth factor activation, mitochondria membrane remodeling, and apoptosis (2-5).A distinctive feature of APP processing compared with other RIPs is the existence of two proteases (␤-and ␣-secretase) that are both capable of executing the first cleavage. Recent studies have shown that ␤-secretase cleavage removes the large extracellular domain of APP, freeing the N terminus of ␤CTF, which is recognized by nicastrin (6). Recruiting ␤CTF to the docking site makes the substrate accessible to the active site of ␥-secretase for hydrolysis. However, the role of nicastrin for substrate binding has been controversial (7-9). Nevertheless, ␤-secretase cleavage converts a latent substrate of ␥-secretase into an active one. Whether ␣-secretase plays a role similar to that of ␤-secretase in the regulation of APP processing by ␥-secretase is unknown. It has been suggested that ␣-secretase competes with ␤-...