Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Chagas, Jair Ribeiro; Martino, Michèle Ferrer-Di; Gauthier, Francis; Lalmanach, Gilles

doi:10.1016/0014-5793(96)00822-8

Cited by 38 publications

(36 citation statements)

References 30 publications

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“…4 ported to be inhibited most strongly by the N-terminal basic segment of the propeptide (41), whereas a lysine residue in the C-terminal region of the propeptide is known to interact with the active site 2 aspartic acid residues in the pepsinogen molecule (43). As for cathepsin B, two separate 5-6-residue peptide segments in the central region of the propeptide and a cysteine residue in one of these segments, which is assumed to interact with the oxyanion hole at the active site of the enzyme, were suggested to be important for the inhibition (14,42). In proproptein convertases, the importance for inhibition was reported for the peptide fragments from the C terminus of each propeptide and especially for the C-terminal seven residues corresponding to the P1-P7 positions relative to the autocatalytic cleavage site (23)(24)(25).…”

Section: Thermal Stabilization Of Agp By Truncated Propeptides and Almentioning

confidence: 99%

“…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%

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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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Section: Thermal Stabilization Of Agp By Truncated Propeptides and Almentioning

confidence: 99%

mentioning

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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“…The relative contribution of each region of cathepsin L propeptide to binding to mature enzyme has been measured by successive truncations of the recombinant propeptide (25,26). We have used a different approach, based on the construction of synthetic peptides overlapping the sequence of the prosegment of cathepsin B, to identify the critical residues interacting with the active site of the mature enzyme (27). But the primary sequences of the proregion of parasite cysteine proteinases differ significantly from those of their mammalian and plant homologues, raising the question of whether the proteolytic activity of parasite proteinases is similarly regulated by their proregion.…”

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confidence: 99%

“…Peptides Pcp24, Pcp25, Pcp26, Pcp27, Pcp32, and Pcp33 were analyzed by reverse phase-HPLC (Brownlee C18 OD 300 column) as indicated above. (27). The peptide HIV1-V3-13 is derived from the V3 loop of Gp120 (29).…”

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confidence: 99%

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

Lalmanach

Lecaille

Chagas

et al. 1998

Journal of Biological Chemistry

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The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in K i value was obtained upon Nterminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.Cysteine proteinases of the papain superfamily have very similar sequences and a common catalytic mechanism; they are found in bacteria, protozoa, plants, and mammals (1, 2). These enzymes play a critical role in the life cycle of protozoan parasites, in host invasion and alteration of the host immune response (3, 4). Parasite cysteine proteinases from the genus Trypanosoma and Plasmodium, have been reported as putative targets for chemotherapeutic inhibitors (5, 6). The main drawback of this strategy is that cysteine proteinases have a broad substrate specificity, which makes it difficult to develop inhibitors that target individual proteinases.Congopain is a cathepsin L-like cysteine proteinase of Trypanosoma congolense, and cruzipain is the equivalent from Trypanosoma cruzi. These protozoan parasites cause bovine trypanosomiase in Africa and Chagas disease in South America respectively (3, 4). Cruzipain (also called cruzain) is a highly mannosylated glycoprotein and an immunodominant antigen (GP57/51) that is among the best characterized of parasitic cysteine proteinases (7-11). The three-dimensional structure of its recombinant central catalytic domain (215 residues) is very similar to those of papain and cathepsin L (11, 12). Congopain differs only slightly from cruzipain in its enzymatic specifici...

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“…23) Chagas et al constructed 10 overlapping 15-mer peptidyl amides spanning the proregion of rat cathepsin B to identify the minimal segments with inhibitory activity toward the mature enzyme. 24) Three synthetic peptidyl amides containing the pentapeptide LCGTV, f(41-45) of rat procathepsin B, inhibited cathepsin B. They also reported that the peptidyl amide, f(36-50) of rat procathepsin B, showed the most potent inhibition and that the Cys-42 region might be essential because alkylation at this residue resulted in rapid proteolytic degradation of the peptide.…”

Section: Discussionmentioning

confidence: 99%

Isolation of Novel Peptides, Cabin-1, -2, -3, and -4, That Inhibit Cathepsin B from a Thermolysin Digest of Human Plasma.

Nakagomi

Fujimura

Maeda

et al. 2002

Biological & Pharmaceutical Bulletin

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Lysosomal cysteine proteases are known to play important roles in intracellular protein degradation 1) and bone resorption.2) They have also been implicated in various pathological processes such as tumor invasion and metastasis, 3) cartilage degradation in arthritis, 4) and muscular dystrophy. 5) Among these lysosomal enzymes, cathepsin B (EC 3.4.22.1) represents a major component that exhibits both endopeptidase and exopeptidase activities. Cathepsin B has also been described as a strong candidate for a prorenin-processing enzyme. 6,7)Recently, cathepsin B inhibitory peptides derived from bcasein such as PFPGPI and GPFPI have been reported by Lee and Lee. 8) Several enzyme inhibitory peptides have also been isolated from enzymatic hydrolysates of proteins. [9][10][11][12][13][14] We previously isolated Acein-1 10) and Acein-2, 11) which are novel peptides that inhibit angiotensin I-converting enzyme (ACE). Acein-1 was isolated from a tryptic hydrolysate of human plasma with the amino acid sequence of Tyr-LeuTyr-Glu-Ile-Ala-Arg, which corresponds to f(138-144) of human serum albumin. Acein-2 (Leu-Ile-Tyr) was isolated from the same hydrolysate, which corresponds to f(518-520) of human a2-macroglobulin. These findings strongly suggest the possibility of isolating some novel cathepsin B inhibitory peptides from enzymatic hydrolysates of proteins.Almost all organisms, including animals, plants, and microorganisms, as well as a large number of synthetic compounds have been examined in the search for bioactive substances as starting materials for medicines.15-18) However, we believe that the human body might be the best resource for such bioactive peptides because such substances would be expected to be less toxic to humans. We have therefore been attempting to discover novel bioactive peptide sequences in human proteins.In this paper, we report the isolation of four novel peptides that inhibit cathepsin B, designated as Cabin-1, -2, -3, and -4, from a thermolysin (EC 3.4.24.4) digest of human plasma.To our knowledge, this is the first report on the isolation of cathepsin B inhibitory peptides from human protein. We also discuss their inhibitory mechanisms based on Lineweaver-Burk plot analysis. MATERIALS AND METHODSInhibitory Assay for Cathepsin B Inhibitory activity of cathepsin B was measured spectrofluorometrically using ZArg-Arg-MCA as the substrate as described by Barret and Kirschke 19) with a slight modification. Cathepsin B (from human placenta, Sigma, St. Louis, MO, U.S.A.) was dissolved in 50 mM sodium acetate buffer (pH 5.0) containing 0.2 M NaCl, 0.5 mM HgCl 2 , and 1 mM EDTA to a final concentration of 0.2 units/ml. A sample (100 ml) was mixed with 400 ml of 0.4 M sodium acetate buffer (pH 5.5) containing 4 mM EDTA, 200 ml of the enzyme solution, and 50 ml of 0.2 M L-cysteine in sodium acetate buffer (pH 5.5) and preincubated for 3 min at 37°C. A 250 ml aliquot of 40 mM Z-ArgArg-MCA (Peptide Institute, Osaka, Japan) in water was added and mixed to start the reaction at 37°C. After precisely 6 min, 1....

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Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

Cited by 38 publications

References 30 publications

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

Isolation of Novel Peptides, Cabin-1, -2, -3, and -4, That Inhibit Cathepsin B from a Thermolysin Digest of Human Plasma.

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