Fluorescent Peptidyl Substrates as an Aid in Studying the Substrate Specificity of Human Prohormone Convertase PC1 and Human Furin and Designing a Potent Irreversible Inhibitor

Jean, François; Boudreault, Alain; Basak, Ajoy; Seidah, Nabil G.; Lazure, Claude

doi:10.1074/jbc.270.33.19225

Cited by 87 publications

(95 citation statements)

References 43 publications

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“…In these studies it was found that only 1 5 2 0 % of prodynorphin was processed at the monobasic site, whereas 80% of the prodynorphin was processed at dibasic sites, suggesting that PC1 prefers processing at dibasic sites within prodynorphin. This is consistent with the data in the present study with shorter fluorescent peptide substrates; PC 1 cleaves peptides that contain basic residues in the P1 and P2 positions more efficiently as compared with the peptides that contain a basic residue only in the PI position (Jean et al, 1995;present study). These results suggest that although PC1 is able to process prodynorphin at monobasic sites, the efficiency of processing at this site is substantially lower than the processing at dibasic sites.…”

Section: Discussionsupporting

confidence: 93%

“…These results suggest that DCE exhibits cleavage site selectivity; this enzyme could be involved in the processing of certain bioactive peptides at sites that require cleavage at type I in addition to cleavage at the type I11 sites. We find that DCE can efficiently process peptides that contain LysLys at the P1 and P2 positions and an Arg at the P4 position; furin and PC1 cannot efficiently process at this site (Jean et al, 1995). Studies with purified PC2 have shown that a Lys-Lys site within the proenkephalin precursor is processed by this enzyme; the efficiency of processing is substantially lower than the efficiency of processing at Lys-Arg sites (Lindberg et al, 1995).…”

Section: Discussionmentioning

confidence: 81%

“…are commonly used to characterize the cleavage specificity of endoproteases (Brenner and Fuller, 1992;Jean et al, 1995). These substrates have a fluorescent group attached to the COOH terminus of the peptide; cleavage at the peptide-AMC bond releases the fluorescent group.…”

Section: Peptide-amino-4-methylcoumarin (Amc) Substratesmentioning

confidence: 99%

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Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

Berman

Juliano

Devi

1999

Journal of Neurochemistry

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Abstract:The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, DnpArg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 81%

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Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

Berman

Juliano

Devi

1999

Journal of Neurochemistry

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“…Furin, PC6B, PC7, and PACE-4 assays were performed in 100 mM HEPES pH 7.5, containing 0.5% Triton X-100 and 1 mM CaCl 2 . PC3 assays were performed as described (Jean et al, 1995). Each enzyme preparation was enzymatically pure based on the absence of PC activity in medium from replicate cells infected with wild-type VV (data not shown).…”

Section: Oocyte Injections and Analysis Of Proteinsmentioning

confidence: 99%

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development

et al. 1998

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“…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 inhibitor 8 (PI8) (23), the turkey ovomucoid third domain (24), and eglin C (25,26).…”

mentioning

confidence: 99%

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

Pullikotil

Vincent

Nichol

et al. 2004

Journal of Biological Chemistry

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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...

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Fluorescent Peptidyl Substrates as an Aid in Studying the Substrate Specificity of Human Prohormone Convertase PC1 and Human Furin and Designing a Potent Irreversible Inhibitor

Cited by 87 publications

References 43 publications

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

Specificity of the Dynorphin‐Processing Endoprotease: Comparison with Prohormone Convertases

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development

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

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