Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state
Presenilin 1 is linked with γ-secretase activity in the detergent solubilized state
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DOI：
10.1073/pnas.110126897
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发表时间：
2000-05-23
影响因子：
11.1
通讯作者：
Gardell, SJ
中科院分区：
文献类型：
--
作者：
Li, YM;Lai, MT;Gardell, SJ
γ-分泌酶是一种膜相关的蛋白酶，它在淀粉样前体蛋白的跨膜区内裂解产生Aβ40和Aβ42两种AP多肽亚型的C末端，本文报道了它的洗涤剂增溶作用和部分特性。用重组底物C100Flag测定溶解的γ-分泌酶的活性，重组底物主要由β-分泌酶裂解位点下游的淀粉样前体蛋白的C末端片段组成。使用特异性识别Aβ40或Aβ42末端的抗体，通过电化学发光检测伽马分泌酶对C100Flag的切割。C100Flag与HeLa细胞膜或洗涤剂增溶的HeLa细胞膜孵育产生Aβ40和Aβ42末端，催化活性、可溶性伽马分泌酶的恢复关键取决于洗涤剂的选择；CHAPSO(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate)而不是Triton X-100是合适的，溶解的伽马分泌酶活性被胃抑素抑制，更有效的是被一种新型的天冬氨酸蛋白酶过渡态类似物抑制，它阻止哺乳动物细胞中Aβ40和Aβ42的形成。凝胶排斥层析显示，溶解的伽马分泌酶活性与早老素1(PS1)相当，表观相对分子质量约为2.0x10(6)。抗PS1抗体免疫共沉淀法从溶解的伽马分泌酶制剂中沉淀伽马分泌酶活性。这些数据表明，伽马分泌酶活性是由含有PS1的大分子复合体催化的。
gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two AP peptide isoforms, A beta 40 and A beta 42, Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the A beta 40 or A beta 42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the A beta 40 and A beta 42 termini, Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable, Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of A beta 40 and A beta 42 in mammalian cells, Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.