Processing of beta-amyloid precursor protein by cathepsin D
Processing of beta-amyloid precursor protein by cathepsin D
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DOI：
10.1074/jbc.271.50.31885
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发表时间：
1996-12-13
影响因子：
4.8
通讯作者：
Cordell, B
中科院分区：
文献类型：
--
作者：
Higaki, J;Catalano, R;Cordell, B
导致从其前体(βAPP)形成β-淀粉样蛋白(βA4)的事件涉及产生βA4的氨基和羧基末端的蛋白分解。负责这些裂解的酶活性分别称为β-分泌酶和伽马-分泌酶，尽管这些酶(S)尚未被鉴定，但由于已知βA4在氨基和羧基末端具有异质性，β-和γ-分泌酶实际上可能是蛋白分解活性的集合，或者可能是具有广泛氨基酸特异性的单个蛋白水解酶。我们研究了组织蛋白酶D在β-APP加工过程中的作用，因为该酶被广泛地认为是伽玛分泌酶候选。用人组织蛋白酶D处理一种跨越β-APP的伽马分泌酶部位的合成肽，导致该底物在ALA(42)-Thr(43)处被切割。为了进一步研究组织蛋白酶D在体外处理较长的重组β-APP底物(156和100个氨基酸的β-APP羧基末端)的能力，用该技术鉴定了这些重组β-APP底物中准确的组织蛋白酶D裂解位点，两种重组底物都被切割在以下位置：Leu(49)-Val(50)，Asp(68)-Ala(69)，Phe(93)-Phe(94)，在可能的伽马分泌酶位点上没有观察到切割：Val(40)-Ile(41)或Ala(42)-Thr(43)，提示组织蛋白酶D不是由这些βA4末端所定义的伽马分泌酶，在βAPP156底物首先变性的条件下，在βA4的氨基末端附近观察到另外两个裂解位点Glu(-3)-Val(-2)和Glu(3)-Phe(4)，表明组织蛋白酶D的裂解受到底物结构完整性的影响，这些结果表明，在体外，组织蛋白酶D不太可能作为伽玛分泌酶发挥作用；然而，这种酶在分子内非淀粉样变部位有效切割βAPP底物的能力可能反映了βAPP分解代谢的作用。
The events leading to the formation of beta-amyloid (beta A4) from its precursor (beta APP) involve proteolytic cleavages that produce the amino and carboxyl termini of beta A4. The enzyme activities responsible for these cleavages have been termed beta- and gamma-secretase, respectively, although these protease(s) have not been identified, Since beta A4 is known to possess heterogeneity at both the amino and carboxyl termini, beta- and gamma-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity.We investigated the role of cathepsin D in the processing of beta APP since this enzyme has been widely proposed as a gamma-secretase candidate. Treatment of a synthetic peptide that spans the gamma-secretase site of beta APP with human cathepsin D resulted in the cleavage of this substrate at Ala(42)-Thr(43). A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant beta APP substrates (156 and 100 amino acids of beta APP carboxyl terminus) in vitro, The precise cathepsin D cleavage sites within these recombinant beta APP substrates were identified using this technique, Both recombinant substrates were cleaved at the following sites: Leu(49)-Val(50), Asp(68)-Ala(69), Phe(93)-Phe(94), No cleavages were observed at putative gamma-secretase sites: Val(40)-Ile(41) or Ala(42)-Thr(43), suggesting that cathepsin D is not gamma-secretase as defined by these beta A4 termini, Under conditions where the beta APP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of beta A4, Glu(-3)-Val(-2) and Glu(3)-Phe(4), were observed, indicating that cathepsin D cleavage of beta APP is influenced by the structural integrity of the substrate, Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as gamma-secretase; however, the ability of this enzyme to efficiently cleave beta APP substrates at nonamyloidogenic sites within the molecule may reflect a role in beta APP catabolism.