Espenshade P J, Cheng D, Goldstein J L, Brown M S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
J Biol Chem. 1999 Aug 6;274(32):22795-804. doi: 10.1074/jbc.274.32.22795.
Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH(2)-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH(2)-terminal signal sequence of S1P generates S1P-A (amino acids 23-1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187-1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P.
位点1蛋白酶（S1P）是一种与枯草杆菌蛋白酶相关的蛋白酶，它在内质网腔中切割固醇调节元件结合蛋白（SREBPs），从而启动一个过程，通过该过程SREBPs转录活性的NH2末端片段从膜上释放出来。在当前实验中，我们将编码表位标记的仓鼠S1P的cDNA转染到HEK-293细胞或缺乏S1P的突变仓鼠细胞中。蛋白酶保护试验表明，大部分S1P位于内质网腔中，由COOH末端跨膜片段锚定。S1P的NH2末端信号序列被切割后产生无活性的S1P-A（氨基酸23 - 1052）。该蛋白通过在B位点的分子内切割而自我激活，产生S1P-B（氨基酸138 - 1052）并释放出一个完整分泌到培养基中的115个氨基酸的前肽。B位点的序列是RSLK，与SREBP-2切割位点的RSVL序列不同。S1P-B在内部RRLL序列处进一步切割产生S1P-C（氨基酸187 - 1052）。突变分析表明，S1P-B和S1P-C都以需要SREBP切割激活蛋白的方式在切割SREBP-2方面具有活性。S1P-C的活性可能是短暂的，因为它似乎被转运到高尔基体，而高尔基体通常不是SREBP-2切割发生的位点。这些数据提供了对一种控制血液和细胞中胆固醇水平的枯草杆菌蛋白酶相关蛋白酶加工过程的初步描述。在一篇配套论文中（Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805 - 22812），我们开发了一种体外测定法来表征纯化的重组S1P的活性。
