Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins
Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins
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DOI：
10.1074/jbc.274.32.22805
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Goldstein, JL
中科院分区：
文献类型：
--
作者：
Cheng, D;Espenshade, PJ;Goldstein, JL
我们描述了一个永久性的中国仓鼠卵巢细胞系转染的cDNA编码的截短形式的网站-1蛋白酶（S1P），分泌到培养基中的酶活性的形式。S1P是一种枯草杆菌蛋白酶样蛋白酶，通常切割固醇调节元件结合蛋白（SREBP）的管腔环。这种切割启动了两步蛋白水解过程，通过该过程，SREBP的NH 2-末端结构域从细胞膜释放以易位到细胞核，在细胞核中它们激活参与胆固醇和脂肪酸的生物合成和摄取的基因的转录。由转染的中国仓鼠卵巢细胞产生的截短S1 P（氨基酸1 - 983）缺乏COOH末端膜锚。与天然S1P一样，这种截短的蛋白质在残基137后经历正常的自催化加工以释放NH2末端前肽，从而产生活性形式，命名为S1P-B。在分泌之前，截短的S1P-B，像天然S1P-B一样，在残基186之后被进一步切割以产生S1P-C，这是培养基中出现的唯一形式。分泌的酶，命名为S1P（983）-C，切割终止于7-氨基-4-甲基-香豆素荧光染料的合成肽。该肽RSLK-MCA对应于产生S1P-B的内部前肽切割位点，如所附论文（Espenshade，P.J.，郑，D.，戈尔茨坦，J.L.，Brown，M. S.（1999），J.Biol.Chem.274，22795 - 22804）。分泌的酶不切割RSVL-MCA，RSVL-MCA是一种对应于SREBP-2中生理切割位点的肽。然而，当RSVL序列包含在对应于SREBP-2管腔环的中心部分的16个残基肽内时，S1P（983）-C确实在该亮氨酸之后切割。S1P（983）-C的催化活性与弗林蛋白酶/激素原转化酶（两种相关蛋白酶）的催化活性不同之处在于其更碱性的最适pH（pH 7 - 8）、其对钙螯合剂的相对抗性以及其在赖氨酸或亮氨酸而不是精氨酸之后裂解的能力。这些数据提供了直接的生物化学证据，即S1P是切割SREBP的蛋白酶，从而起到控制动物细胞中脂质生物合成和摄取的作用。
We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH2-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1-983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH2-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells.