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首页> 外文期刊>Biochemistry >Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates
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Proteolytic activation of recombinant pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic substrates

机译：用新的荧光底物研究了重组蛋白原蛋白2（pro-β-分泌酶）的蛋白水解激活。

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Memapsin 2 (beta-secretase), a membrane-anchored aspartic protease, is involved in the cleavage of beta-amyloid precursor protein to form beta-amyloid peptide. The primary structure of memapsin 3 suggests that it is synthesized in vivo as pro-memapsin 2 and converted to memapsin 2 by an activating protease [Lin et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1456-1460]. To simulate this activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several proteases with trypsin-like specificity. Clostripain, kallikrein, and trypsin increased the activity of pro-memapsin 2, Clostripain activation was accompanied by the cleavage of the pro region to form mainly two activation products, Leu(30p)- and Gly(45p)-memapsin 2, Another activation product, Leu(28p)-memapsin 2, was also purified. Kinetics of the activated memapsin 2 were compared with pro-memapsin 2 using two new fluorogenic substrates, Arg-Glu(5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic acid (EDANS))-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys (4-(4-dimethylaminophenylazo)benzoic acid (DABCYL))-Arg and (7-methoxycoumarin-4-yl)acetyl (MCA))-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys(2,4-dinitrophenyl (DNP)). These results establish that the activity of promemapsin 2 stems from a part-time and reversible uncovering of its active site by its pro region. Proteolytic removal of part of the pro-peptide at Leu(28p) or Gly(45p), which diminishes the affinity of the shortened pro-peptide to the active site, results in activated memapsin 2. These results also suggest that Glu(33p)- memapsin 2 observed in the cells expressing this enzyme [Vassar et al, (1999) Science 286, 735-741; Yan et al. (1999) Nature 402, 533-537] is an active intermediate of in vivo activation, or that the peptide Glu(33p)-Arg(44p) may serve a regulatory role. [References: 25]
机译：Memapsin 2（β-分泌酶）是一种膜锚定的天冬氨酸蛋白酶，参与β-淀粉样蛋白前体蛋白的裂解，形成β-淀粉样蛋白肽。 memapsin 3的一级结构表明它在体内合成为pro-memapsin 2，并通过活化蛋白酶转化为memapsin 2 [Lin等。 （2000年）过程。 Natl。学院科学[U.S.A. 97，1456-1460]。为了模拟这种激活机制并为动力学/特异性研究产生稳定的成熟美美皮素2，我们研究了几种具有胰蛋白酶样特异性的蛋白酶对重组美美白素2的激活。 Clostripain，激肽释放酶和胰蛋白酶增加了pro-memapsin 2的活性，clostripain激活伴随着pro区域的裂解，主要形成两个激活产物Leu（30p）-和Gly（45p）-memapsin 2，另一个激活产物。还纯化了Leu（28p）-memapsin 2。使用两种新的荧光底物Arg-Glu（5-[（（2-氨基乙基）氨基]-萘-1-磺酸（EDANS））-Glu-Val-Asn将活化的memapsin 2和pro-memapsin 2的动力学进行了比较-Leu-Asp-Ala-Glu-Phe-Lys（4-（4-二甲基氨基苯基偶氮）苯甲酸（DABCYL））-Arg和（7-甲氧基香豆素-4-基）乙酰基（MCA））-Ser-Glu-Val- Asn-Leu-Asp-Ala-Glu-Phe-Lys（2,4-二硝基苯基（DNP））。这些结果表明，promemapsin 2的活性源于其pro区域的部分时间和可逆地发现其活性位点。通过蛋白水解去除Leu（28p）或Gly（45p）处的部分原肽，从而减少了缩短的原肽对活性位点的亲和力，从而激活了memapsin2。这些结果也表明，Glu（33p） -在表达该酶的细胞中观察到的美皮素2 [Vassar等人，（1999）Science 286，735-741; Vesar等人，（1999）Science 286，735-741。严等。 （1999）Nature 402，533-537]是体内激活的活性中间体，或者说肽Glu（33p）-Arg（44p）可能起调节作用。 [参考：25]

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