Substrate Cleavage Analysis of Furin and Related Proprotein Convertases

Abstract

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.

Proprotein Convertase Subtilisin/Kexin Type 7 (PCSK7) Is Essential for the Zebrafish Development and Bioavailability of Transforming Growth Factor β1a (TGFβ1a)

“…Zebrafish homologs for the neuroendocrine system-specific PCSK1 and PCSK2 were particularly highly expressed in the fish neural tissues, and the homologs of previously reported ubiquitous enzymes, such as FURIN, PCSK5, and PCSK7, were found to be relatively widely expressed similarly to earlier reports using mammals (17,18,47). PCSK7 shares several common substrate molecules with PCSK5 and FURIN at least in in vitro analyses (48). We found that pcsk7 was co-expressed in different tissues and developing fish together with other PCSK enzymes that have been reported FIGURE 9.…”

The (pro)renin receptor is cleaved by ADAM19 in the Golgi leading to its secretion into extracellular space

“…Remacle et al reported that RXKR or RXRR, but not RKXR, in (P)RR strongly contributes to the furin cleaving sites. 20 Hence, furin may not be pivotal in the processing of (P)RR. Furthermore, M8-9, which is associated with the V-ATPase, corresponds to carboxyl-terminal amino acid sequences of (P)RR.…”

Post-Endoplasmic Reticulum Rescue of Unstable MHC Class I Requires Proprotein Convertase PC7

“…We decidedtoanalyzethe contribution of PC7toMHCI Agprocessing by a cell biologic assay, in which we used a defined 15-mer peptide of EBV glycoprotein B (EBVgB, GP110), which is a well-known target substrate of pPCs (26,27). The EBVgB-peptide gB427-441 (LRRRRR↓DAGNATTPV, sequence in single-letter code) contains the pPC-cleavage motif-(R-X-K/R-R↓) confirmed by ProP 1.0 analysis (28)-conserved among most herpes virus gBs (29) (positions 3-6, indicated in bold letters and by a down arrow at the cleavage site) as well as a nonameric peptide (downstream the cleavage site, positions 7-15, indicated by italics) with sequence properties of a high-affinity HLA-B51 binding ligand (SYFPEITHI 1.0-score: 24) (30).…”