Molecular mechanisms of γ-cleavage for the generation of amyloid β-protein

为了了解早老素依赖的β分泌酶降解APP(生成Aγ)的分子机制，研究了APP的γ-和γ‘(ε-裂解之间的关系。APP的γ‘-裂解位点附近的氨基酸取代改变了γ’-裂解位点和γ-裂解位点，导致Aβ42产物的比例改变。研究了家族性阿尔茨海默病相关APP和早老素基因突变对γ和γ‘裂解位点的影响。这里检测的所有突变都改变了γ裂解位点，并增加了Aβ42产生的比例。这些突变还改变了γ‘-裂解位点，并导致CTFγ49-99产生的比例增加。虽然观察到Aβ42和CTPγ49-99的产生之间存在潜在的联系，但Aβ40和Aβ42的比例并不能真实地反映γ50-99和49-99的比例。这些结果表明γ-和Y‘-裂解之间存在密切的相互关系。为了评估y‘-切割先于γ-切割的假设，我们试图找到一个中间产物，Aβ物种长于Aβ42/43。利用新开发的尿素凝胶系统和N端特异的Aβ单抗，在不同的细胞类型中明确地鉴定出较长的AβS、Aβ1-43、Aβ1-45、Aβ1-46和Aβ1-48。对那些较长的AβS的精确分析表明，APP的γ-和‘γ’-裂解位点之间存在多个依赖于γ分泌酶的裂解位点，并且APP在γ‘-裂解产生的羧基末端每三个残基上连续裂解。

To understand molecular mechanisms of the proteolytic processing of APP (generation of Aβ) by presenilin-dependent γ-secretase, the relationship between γ-and γ' (ε)-cleavages of APP was investigated.1. Amino acid substitutions in the vicinity of γ'-cleavage sites of APP altered γ'-cleavage sites as well as γ-cleavage sites, resulting in altered proportions of Aβ42 production.2. Effects of familial Alzheimer's disease-associated mutations of APP and presenilins on γ-and γ'-cleavage sites were investigated. All the mutations examined here altered γ-cleavage sites and increased the proportion of Aβ42 production. Those mutations altered also γ'-cleavage sites and caused an increase in the proportion of CTFγ49-99 production. Although a potential link between the production of Aβ42 and CTPγ49-99 was observed, the proportion of Aβ4O and Aβ42 did not faithftully reflect that of CTFγ50-99 and 49-99.These results indicate an intimate interrelation between γ-and y '-cleavages.3. To evaluate the hypothesis that y '-cleavage precedes γ-cleavage, we sought to find an intermediate product, Aβ species longer than Aβ42/43. Using newly developed urea gel systems and an N-end-specific Aβ monoclonal antibody, the longer Aβs, Aβ1-43, Aβ1-45, Aβ1-46, and Aβ1-48, were identified unambiguously within the various cell types. The precise analysis of those longer Aβs suggests that multiple γ-secretase-dependent cleavage sites are present between γ-and 'γ'-cleavage sites of APP and that APP is cleaved successively at every three residues from the carboxyl-terminus generated by γ'-cleavage.