Cheng D, Espenshade P J, Slaughter C A, Jaen J C, Brown M S, Goldstein J L
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
J Biol Chem. 1999 Aug 6;274(32):22805-12. doi: 10.1074/jbc.274.32.22805.
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 NH(2)-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 NH(2)-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.
我们描述了一种中国仓鼠卵巢细胞的永久细胞系，该细胞系转染了编码截短形式的位点-1蛋白酶（S1P）的cDNA，截短的S1P以酶活性形式分泌到培养基中。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（SREBPs）的腔内环。这种切割启动了两步蛋白水解过程，通过该过程，SREBPs的NH(2)-末端结构域从细胞膜释放出来，转运到细胞核，在那里它们激活参与胆固醇和脂肪酸生物合成及摄取的基因的转录。转染的中国仓鼠卵巢细胞产生的截短S1P（氨基酸1-983）缺乏COOH-末端膜锚定结构。与天然S1P一样，这种截短蛋白在第137位残基后经历正常的自催化加工，释放出NH(2)-末端前肽，从而产生一种活性形式，称为S1P-B。在分泌之前，截短的S1P-B与天然S1P-B一样，在第186位残基后进一步切割，产生S1P-C，这是培养基中出现的唯一形式。分泌的酶称为S1P(983)-C，它能切割一种以7-氨基-4-甲基香豆素荧光染料结尾的合成肽。这种肽RSLK-MCA对应于随附论文（Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804）中所述产生S1P-B的内部前肽切割位点。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腔内环中央部分的16个残基肽中时，S1P(983)-C确实会在该亮氨酸之后切割。S1P(983)-C的催化活性与其相关蛋白酶弗林蛋白酶/激素原转化酶的催化活性不同，其最适pH值更偏碱性（pH 7-8），对钙螯合剂相对抗性较强，并且能够在赖氨酸或亮氨酸之后而不是精氨酸之后切割。这些数据提供了直接的生化证据，表明S1P是切割SREBPs的蛋白酶，从而在动物细胞中发挥控制脂质生物合成和摄取的作用。
