The Proprotein Convertase SKI-1/S1P

Abstract

Subtilisin kexin isozyme-1 (SKI-1) represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues. It is synthesized as an inactive precursor that undergoes three sequential autocatalytic processing steps of its N-terminal prosegment and an ectodomain shedding at a site near the transmembrane domain. The various cellular functions of SKI-1 emphasize the need to understand the sites of its activation and shedding. We have previously shown that SKI-1 undergoes autocatalytic shedding at the sequence KHQKLL 953 2, resulting in a membrane-bound stump called St-1 (amino acids 954 -1052). However, little is known about the cellular localization of SKI-1 or its shed forms. In the present study, we have further identified a smaller C-terminal fragment St-2 generated closer to the transmembrane domain. By sequencing and mass spectrometric analysis, the start site and the molecular mass of St-2 were determined. Site-directed mutagenesis revealed the critical amino acid involved in this novel process. Mutation of Met 990 to M990A, M990I, and M990L failed to generate St-2, suggesting an internal alternate translation event at Met 990 , as confirmed by an in vitro transcription/translation assay. Confocal microscopy defined the subcellular localization of SKI-1 and its fragments. The data show that most of membrane-bound SKI-1 and its stumps St-1 and St-2 localize to the Golgi and can enter the endosomal/lysosomal compartments but do not sort to the cell surface. Deletion studies showed that the transmembrane domain of SKI-1 determines its trafficking. Finally, rSt-1 and rSt-2 seem to affect the processing of ATF6 by SKI-1, but cellular stress does not regulate the production of St-2.Several secretory proteins are synthesized as inactive precursors, which when converted to their mature forms by proteolytic enzymes generate a large diversity of bioactive proteins and peptides. The nine-member family of the proprotein convertases (PCs) 2 participates actively in the generation of such molecular diversity (1-3). There are seven basic amino acidspecific kexin-like mammalian proprotein convertases that cleave various precursors at the general consensus motif (K/R)X n (K/R)2, where n ϭ 0, 2, 4, or 6, and X represents any amino acid. The eighth member is the pyrolysin-like subtilisin kexin isozyme-1 (SKI-1) (4), also known as site-1 protease (S1P) (5). It cleaves substrates at the consensus motif (R/K)X-(hydrophobic)-X2, where X is variable (6). The last member PCSK9 cleaves the sequence VFAQ 152 2 within its prosegment (7). SKI-1 represents the first mammalian member of secretory subtilisin-like processing enzymes that cleaves after nonbasic residues (2, 3). The ubiquitously expressed convertase SKI-1 (4) regulates the synthesis of cholesterol and fatty acids and their metabolism, through the processing of the membrane-bound transcription factors sterol regulatory element-binding proteins (8, 9). It also regulates endoplasmic reticulum (ER)-stress response through cleavag...

Site-1 protease regulates skeletal stem cell population and osteogenic differentiation in mice

“…S1P ablation in the Osx lineage, however, did not affect adipogenesis, indicating that this ablation did not interfere with other progenitor functions in the bone marrow. As S1P is not a secreted protein ( Pullikotil et al, 2007 ), this observation may suggest that S1P ablation in the Osx lineage in the bone marrow does not cause non-cell autonomous mutational effects, in agreement with mosaic analysis in zebrafish ( Schlombs et al, 2003 ). How S1P maintains the SSC population is not known as we have not yet identified a molecular target, but our studies clearly indicate a requirement for S1P in the Osx lineage for normal bone development.…”

Envelope Glycoprotein of Arenaviruses

“…Moreover, the data at hand indicate that viral and cellular substrates of SKI-1/S1P are processed in distinct subcellular compartments. In uninfected cells, membrane-associated SKI-1/S1P is found predominantly in the early Golgi where cellular SKI-1/S1P substrates are cleaved [103]. In contrast, LASV GPC is cleaved early in the secretory pathway and LCMV GPC was shown to be processed in a late Golgi or post-Golgi compartment [86].…”

Luminescent reporter cells enable the identification of broad‐spectrum antivirals against emerging viruses

“…Ideally, library screening would result in the identification of selective inhibitors, which primarily block virus GP cleavage. This is conceivable for arenaviruses, since the major cellular SKI‐1/S1P substrates SREBP, N ‐acetylglucosamine‐1‐phosphate transferase subunits alpha and beta, and the transcription factor ATF‐6, are cleaved in the central Golgi stacks 46 while LASV GP is cleaved in the ER, 34 and LCMV GP in the trans ‐Golgi 33,35 . Given that all intermediate SKI‐1/S1P forms generated by auto‐processing during SKI‐1/S1P maturation are enzymatically active, 16,17 they might display compartment specific, differential substrate specificities, potentially allowing selective inhibition of viral GP cleavage with a favorable side effect profile.…”