Synthetic peptides derived from the prosegments of proprotein convertase 1/3 and furin are potent inhibitors of both enzymes

Basak, Ajoy; Lazure, Claude

doi:10.1042/bj20030120

Cited by 37 publications

(41 citation statements)

References 42 publications

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“…These roles, however, have not yet been fully understood, and further studies on various proteins are necessary. As for the inhibitory properties of the propeptides of peptidases, there are numbers of reports describing their inhibition profiles and type of inhibition (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). However, only a few attempts have been made so far to identify critical sequences and/or residues in the propeptide important for inhibition of the corresponding mature peptidase, and much of the inhibition mechanisms, the structural determinants in the propeptide, and the sites of binding involved in the inhibition remains to be established.…”

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confidence: 99%

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Kubota

Nishii

Kojima

et al. 2005

Journal of Biological Chemistry

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Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced byAspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala 1 -Asn 41 ) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (K i ‫؍‬ 27 nM at pH 4. Propeptides are known to be present in many protein precursors mostly at their N termini and are assumed to play various roles in the proteins such as inhibition of protein functions, stabilization of precursor structures, promotion of polypeptide folding, and/or intercellular protein sorting and targeting (1-6). These roles, however, have not yet been fully understood, and further studies on various proteins are necessary. As for the inhibitory properties of the propeptides of peptidases, there are numbers of reports describing their inhibition profiles and type of inhibition (7-25). However, only a few attempts have been made so far to identify critical sequences and/or residues in the propeptide important for inhibition of the corresponding mature peptidase, and much of the inhibition mechanisms, the structural determinants in the propeptide, and the sites of binding involved in the inhibition remains to be established. On the other hand, there are few studies that analyzed the effects of propeptide and its truncated fragments on the stability of the mature enzyme to identify critical sequences and/or residues contributing to the enzyme stability.Aspergilloglutamic peptidase (AGP 1 ; MEROPS ID: G01.002; formerly called aspergillopepsin II) is a pepstatin-insensitive acid endopeptidase produced by the fungus Aspergillus niger var. macrosporus (26). Like scytalidoglutamic peptidase (or eqolisin; MEROPS ID: G01.001; formerly called scytalidopepsin B) (27, 28), AGP also belongs to the newly established family of glutamic peptidases (i.e. peptidase family G1). The enzyme has two essential residues, a glutamic acid and a glutamine residue, at the active site (29 -32), which is thought to form a catalytic dyad, and is not inhibited by any of the known inhibitors for aspartic, cysteine, metallo-, and serine peptidases. The enzyme is synthesized as a 282-residue preproenzyme, and after removal of the 18-residue putative signal peptide, the proform is converted autocatalytically to the twochain mature enzyme (composed of a 39-residue light chain and a 173-residue heavy chain) by liberation of the putative 41-residue propeptide (Ala 1 -Asn 41 ) and the 11-residue intervening peptide...

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confidence: 99%

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Kubota

Nishii

Kojima

et al. 2005

Journal of Biological Chemistry

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“…Furthermore, we and others have previously shown that synthetic peptides of various lengths did not exhibit any significant selectivity (14,17,19) nor did the isolated propeptides when assayed against a variety of convertases (19). However, single amino acid substitutions were shown to exhibit a profound effect on both inhibition and activation of convertases.…”

Section: Resultsmentioning

confidence: 99%

“…Mechanism of Inhibition of PC1/3 Propeptide Mutants toward mPC1/3 and Human Furin-Synthetic inhibitory peptides against convertases, engineered from the COOH-terminal part, presented a competitive pattern of inhibition supporting the fact that this portion of the propeptide interacts directly with the active site (14,19). Upon elongating these peptides toward the NH 2 terminus, a change in inhibitory behavior was observed, because the initial competitive inhibition changed with the length of the peptide to mixed-inhibition and even FIGURE 1.…”

Section: Resultsmentioning

confidence: 99%

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Single Amino Acid Substitution in the PC1/3 Propeptide Can Induce Significant Modifications of Its Inhibitory Profile toward Its Cognate Enzyme

Rabah

Gauthier

Wilkes

et al. 2006

Journal of Biological Chemistry

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The proprotein convertase PC1/3 is synthesized as a large precursor that undergoes proteolytic processing of the signal peptide, the propeptide and ultimately the COOH-terminal tail, to generate the mature form. The propeptide is essential for protease folding, and, although cleaved by an autocatalytic process, it remains associated with the mature form acting as an auto-inhibitor of PC1/3. To further assess the role of certain residues in its interaction with its cognate enzyme, we performed an alanine scan on two PC1/3 propeptide potential cleavable sites ( 50 RRSRR 54 and 61 KR 62 ) and an acidic region 65 DDD 67 conserved among species. Upon incubation with PC1/3, the ensuing peptides exhibit equal inhibitory potency, lower potency, or higher potency than the wild-type propeptide. The K i values calculated varied between 0.15 and 16.5 nM. All but one mutant exhibited a tight binding behavior. To examine the specificity of mutants, we studied their reactivity toward furin, a closely related convertase. The mutation of certain residues also affects the inhibition behavior toward furin yielding propeptides exhibiting K i ranging from 0.2 to 24 nM. Mutant propeptides exhibited against each enzyme either different mode of inhibition, enhanced selectivity in the order of 40-fold for one enzyme, or high potency with no discrimination. Hence, we demonstrate through single amino acid substitution that it is feasible to modify the inhibitory behavior of propeptides toward convertases in such a way as to increase or decrease their potency, modify their inhibitory mechanisms, as well as increase their selectivity.One of the most common methods used by cells to diversify the pool of their biologically active molecules is protein processing. Indeed, numerous secreted proteins are synthesized first as an inactive precursor, which is rendered biologically active upon cleavage at clusters of basic residues. Members of a family of proteins named proprotein convertases (PCs) 3 primarily perform this cleavage. To date, seven members were described: furin, PC1/3, PC2, PACE4, PC4, PC5/6, and PC7/ PC8/lymphoma proprotein convertase. Some of them are ubiquitously expressed such as furin, PACE4, and PC7, whereas others exhibit a more restricted expression pattern such as PC1/3 and PC2, which are solely present in endocrine and neuroendocrine tissues, and PC4, which is expressed only in germ cells. However, all of them belong to the larger family of serine proteases and are structurally related to bacterial subtilisin and yeast kexin (reviewed in Ref. 1). In terms of biological activities, numerous transfection experiments using recombinant enzymes and substrates, generation of knock-out animals as well as human cases of convertase deficiency pointed out the importance of convertases in crucial biological processes such as patterning during embryogenesis, angiogenesis, prohormone processing, tissue remodeling, and complement activation (2). Furthermore, some members are also implicated in many disease states, because they are able to...

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“…Hence our biosynthetic data and modelling support this concept, whereby a zymogen with a pro-region existing in a state of low affinity for its catalytic domain retains intrinsic enzymic activity. Interestingly, recent results obtained on the inhibitory properties of peptides derived from the pro-regions of PC1 and furin have demonstrated how residues His-66 and Phe-67 may well contribute to the affinity of pro-regions for the catalytic domains [40].…”

Section: Discussionmentioning

confidence: 99%

Identification of furin pro-region determinants involved in folding and activation

Bissonnette

Charest

Longpré

et al. 2004

Biochemical Journal

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The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the transGolgi network. To identify residues that are important for proregion function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's proregion.

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Synthetic peptides derived from the prosegments of proprotein convertase 1/3 and furin are potent inhibitors of both enzymes

Cited by 37 publications

References 42 publications

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Single Amino Acid Substitution in the PC1/3 Propeptide Can Induce Significant Modifications of Its Inhibitory Profile toward Its Cognate Enzyme

Identification of furin pro-region determinants involved in folding and activation

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