Autocatalytic Processing of Site-1 Protease Removes Propeptide and Permits Cleavage of Sterol Regulatory Element-binding Proteins

Abstract

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.Cholesterol metabolism in animal cells is controlled by the sterol-regulated proteolysis of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) (1).1 The sterol-regulated reaction is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves SREBPs in a hydrophilic loop that projects into the lumen of the endoplasmic reticulum (ER) and other organelles (2). An understanding of the mechanism of regulation of S1P is essential if we are to understand how animals control the cholesterol content of cells and blood. SREBPs are a family of three proteins, each of ϳ1150 amino acids in length. After synthesis, each SREBP is inserted into the membranes of the ER and nuclear envelope in a hairpin orientation (1). The NH 2 -terminal segment of ϳ480 amino acids is a transcription factor of the basic helix-loop-helix-leucine zipper family that projects into the cytoplasm. The middle segment of ϳ80 amino acids consists of two membrane-spanning helices separated by a luminal hydrophilic loop of ϳ30 amino acids. The COOH-terminal segment of ϳ590 amino acids extends into the cytoplasm, where it forms a complex with the COOH-terminal segment of a membrane-bound regulatory protein designated SREBP-c...

The Lassa virus glycoprotein precursor GP-C is proteolytically processed by subtilase SKI-1/S1P

“…The consensus sequence of Lassa virus glycoprotein was found to be homologous to the recognition motif of the recently discovered SKI-1͞ S1P protease belonging to the pyrolysin branch of subtilases. This enzyme has been identified in human, rat, mouse, and hamster cells, with the human and the hamster form showing 97% identity (11)(12)(13). In the present study, we have identified SKI-1͞S1P as the cleavage enzyme of the Lassa virus glycoprotein.…”

Development of Protein-based Inhibitors of the Proprotein of Convertase SKI-1/S1P

“…Cellular localization experiments revealed that SKI-1 is sorted to the cis/medial Golgi (5,37,63), suggesting that it is poised to process its cognate precursors therein, SREBPs (39), ATF6 (41), Luman (42), and proBDNF (5). Whereas the modified serpin ␣ 1 -PDX (20,21) and the PC-prosegments (31) inhibit the basic amino acid-specific PCs within the constitutive secretory pathway, no specific-inhibitor of SKI-1 is yet known.…”

“…1). The absence of a P4 Arg should block the B-site (7,37) but could still allow the BЈ to interact with the SKI-1 catalytic pocket. It should be noted that the R134E mutant of the full-length SKI-1 is ϳ50% less processed into the BЈ/B and C forms than the wild-type sequence (7).…”

“…The signal peptidase cleavage generates an A form (amino acids ) that is subsequently autocatalytically cleaved in the endoplasmic reticulum (ER) at two alternate BЈ and B sites: RKVF 133 2 (SKI-1-(134 -1052)) and RKVFRSLK 137 2 (SKI-1-(138 -1052)), respectively. The latter products are then transported to the cis/medial Golgi whereupon they are further autocatalytically processed into a C-form at RRLL 186 2, generating SKI-1-(187-1052) (5,7,35,37).…”

Genomic structure and chromosomal mapping of the human Site-1 protease (S1P) gene

“…1B indicates. Exon 1 encodes the 5Ј non- translated region; part of exon 2 encodes the 22 NH2-terminal amino acids that constitute a signal sequence; the remainder of exon 2 and all of exon 3 encode the propeptide sequence that is synthesized as an inactive membrane-bound precursor and is self-activated by intramolecular cleavage to produce active enzyme Espenshade et al 1999;Sakai et al 1998a). Exons 5-10 encode the subtilisin-homology domain, whose aspartate 218, histidine 249, and serine 414 residues are required for the catalytic activity of S1P (Sakai et al 1998a).…”