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

Abstract

Processing of membrane-bound transcription factors such as sterol regulatory element-binding proteins (SREBPs) and the ER-stress response factor ATF6, and glycoproteins of some hemorrhagic fever viruses are initiated by the proprotein convertase SKI-1/S1P. So far, no cellular protein-based inhibitor of the hydrophobicamino acid specific SKI-1 is known. The prosegment of the basic-amino acid specific convertases (e.g. furin and PC5) or ␣ 1 -PDX, a variant of ␣ 1 -antitrypsin (␣ 1 -AT) exhibiting an RIPR 358 sequence at the reactive site loop, were shown to potently inhibit these secretory proteinases. Accordingly, we tested the SKI-1-inhibitory potential of various point mutants of either the 198 amino acid preprosegment of SKI-1-(1-198) or ␣ 1 -AT. Transient transfections data showed that, out of numerous mutants studied, the R134E prosegment mutant or the ␣ 1 -AT reactive site loop variants RRVL 358 , RRYL 358 and RRIL 358 are the best specific cellular inhibitors of SKI-1. The observed inhibition of the processing of endogenous SREBP-2, exogenous ATF6 and a PDGF-A (RRLL 86 ) variant were >55% and reach ϳ80% in stable transfectants. We also show that SKI-1 forms SDS-stable complexes with these ␣ 1 -AT variants, but not with wild-type ␣ 1 -AT or ␣ 1 -PDX. Finally, these inhibitors were also shown to affect the processing and stability of the Crimean-Congo hemorrhagic fever virus glycoprotein.Proteins and peptides that are biologically active are often generated by intracellular limited proteolysis of inactive precursors. The mammalian proprotein convertases (PCs) 1 of the secretory pathway are calcium-dependent serine proteinases related to bacterial subtilisin 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 is any amino acid (1-3). The PC family counts seven basic amino acid-specific kexin-like convertases: furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7/LPC (4). The eighth member is the recently discovered pyrolysin-like SKI-1/S1P that cleaves at the consensus motif (R/K)X(hydrophobic)Z2, where Z is variable (5), while the last member (6, 7) NARC-1 cleaves the sequence VFAQ 153 2 in its prosegment (8, 9). 2 More PCs contain an N-terminal signal sequence, followed by a prosegment, a catalytic domain and a P-domain. In addition, PCs possess a C-terminal segment that varies between the different members. The critical role of PCs in the proteolytic maturation of multiple proproteins, their implication in various pathologies (1, 10, 11), and their unidentified specific and/or redundant functions, make them attractive targets for the development of potent and selective inhibitors. The various successful approaches include: active site-directed chloromethyl ketone inhibitors (12, 13), reversible peptide-based inhibitors (14 -17), plant derivatives (18), and several engineered variants of protein-based inhibitors that possess a furin-like motif. These include ␣2-macroglobulin (␣2-MF) (19), ␣ 1 -antitrypsin (␣ 1 -AT) Portland (␣ 1 -PDX) (20 -22), proteinase i...

Site-directed Mutagenesis, Proteolytic Cleavage, and Activation of Human Proheparanase

“…Cell lysates were incubated (24 h, 37°C, pH 5.8) with 35 Slabeled ECM. The medium was centrifuged, and the supernatant containing the sulfate-labeled degradation fragments was analyzed by gel filtration on a Sepharose CL-6B column (0.9 ϫ 30 cm).…”

“…We have also substituted the conserved Arg 153 with glutamine, assuming that it may constitute a potential RXXX2 recognition motif for a subtilisin/kexin isozyme-1 convertase (33)(34)(35). Again, the R153A mutant (mutation #10) was found to be normally processed and enzymatically active (Fig.…”

“…Two adjacent lysines, Lys 158 and Lys 159 , found at :QQ); lysines 107 and 108 were replaced by glutamine (f, 107 KK 108 :QQ), aspartic acid 105 and proline 106 were replaced by glutamine (•), or 6 (Ⅺ, ⌬STFEER), and 11 (᭜, ⌬STFEERSYWQS) amino acids were deleted. Cell lysates were incubated with 35 S-labeled ECM and tested for heparanase activity by gel filtration (Sepharose 6B) analysis of released sulfate-labeled material, as described under "Experimental Procedures." Jar cells transfected with native proheparanase or each of the mutated constructs exhibited the same heparanase enzymatic activity.…”

Regulation of the stepwise proteolytic cleavage and secretion of PDGF-B by the proprotein convertases

“…The data demonstrated that PCs only process proPDGF-B at the RGRR 81 k site in HK293 cells (Figure 2). Furthermore, the expression of SKI-1 in LoVo-C5 cells did not enhance processing (Figure 2), nor was the C-terminal processing PDGF-B abrogated in SKI-1-deficient CHO cells (Pullikotil et al, 2004) (not shown). Since NARC-1/PCSK9 is not expressed in HK293 cells , the above results rule out the implication of any member of the PC-family in this C-terminal processing event.…”

Inhibition of Proprotein Convertase SKI-1 Blocks Transcription of Key Extracellular Matrix Genes Regulating Osteoblastic Mineralization

“…The culture media contained 145-kDa SKI-1 precursor and a 50-kDa fragment band. Due to the specificity of the antibody and the structure of SKI-1 (31,32,38,39), we assume that all forms detected (145, 105, 70, and 50 kDa) contain the transmembrane sequence and are associated with membranes or vesicles released from cells. Interestingly, the amount of 50-kDa band in the media was found to be lower in mineralized cultures, indicating that breakdown of SKI-1 may be increased under non-mineralizing conditions (Fig.…”