Activation processing of cathepsin H impairs recognition by its propeptide (original) (raw)
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FEBS Letters, 1996
Ten overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues lp-60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p-45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p-50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Kt = 2 ltM) using a longer peptide (26p-50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Kl values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p-G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PBll at the Lys-40p-Leu-41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides.
Potency and Selectivity of the Cathepsin L Propeptide as an Inhibitor of Cysteine Proteases †
Biochemistry, 1996
The cathepsin L propeptide (phcl-2) was expressed in Saccharomyces cereVisiae using a human procathepsin L/R-factor fusion construct containing a stop codon at position -1 (the C-terminal amino acid of the proregion). Since the yield after purification was very low, the cathepsin L propeptide was also obtained by an alternate procedure through controlled processing of an inactive mutant of procathepsin L (Cys25Ser/Thr110Ala) expressed in Pichia pastoris, by small amounts of cathepsin L. The peptide resulting from the cleavage of the proenzyme (phcl-1) was then purified by HPLC. The purified propeptides were characterized by N-terminal sequencing and mass spectrometry and correspond to incomplete forms of the proregion (87 and 81 aa for phcl-1 and phcl-2 respectively, compared to 96 aa for the complete cathepsin L propeptide). The two peptides were found to be potent and selective inhibitors of cathepsin L at pH 5.5, with K i values of 0.088 nM for phcl-1 and 0.66 nM for phcl-2. The K i for inhibition of cathepsin S was much higher (44.6 nM with phcl-1), and no inhibition of cathepsin B or papain could be detected at up to 1 µM of the propeptide. The inhibitory activity was also found to be strongly pHdependent. Two synthetic peptides of 75 and 44 aa corresponding to N-terminal truncated versions of the propeptide were also prepared by solid phase synthesis and displayed K i values of 11 nM and 2900 nM, respectively, against cathepsin L. The data obtained for the 4 propeptide derivatives of various lengths indicate that the first 20 residues in the N-terminal region of the propeptide are more important for inhibition than the C-terminal region which contributes little to the overall inhibitory activity. † NRCC Publication No. 39920. Supported by postdoctoral fellowships from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil (E.C.), as well as NATO and the Ministère
The Inhibition of Cathepsin S by its Propeptide - Specificity and Mechanism of Action
European Journal of Biochemistry, 1997
The interaction of human recombinant full-length cathepsin S propeptide (amino acids 16-114) with mature cysteine proteinases was studied with respect to selectivity and pH dependence. The inhibitory capacity was tested towards mature human recombinant cathepsin S, purified cathepsin L from rat and Paramecium tetraurelia, rat cathepsin B, human cathepsin H, and papain. The propeptide of cathepsin S strongly inhibited cathepsin S (K, = 0.27 nM) and the two cathepsin L species (Kl = 0.36 nM) at neutral pH. Papain, and to a minor extent cathepsin H, hydrolyzed the propeptide of cathepsin S, leading to competition with the hydrolysis of the fluorogenic substrates in the respective assays. Cathepsin B activity was nearly unaffected up to micromolar propeptide concentrations in the assay. The inhibition of cathepsin-L-like peptidases was diminished with decreasing pH, probably due to dramatic changes in the conformation of the propeptide. This assumption was supported by far-ultraviolet CD spectroscopy and by the finding of rapid hydrolysis of the cathepsin S propeptide by cathepsin L at pH values less than 5.5.
Biochemistry, 2001
Several new cysteine proteases of the papain family have been discovered in the past few years. To help in the assignment of physiological roles and in the design of specific inhibitors, a clear picture of the specificities of these enzymes is needed. One of these novel enzymes, cathepsin X, displays a unique specificity, cleaving single amino acid residues at the C-terminus of substrates very efficiently. In this study, the carboxypeptidase activities and substrate specificity of cathepsins X and B have been investigated in detail and compared. Using quenched fluorogenic substrates and HPLC measurements, it was shown that cathepsin X preferentially cleaves substrates through a monopeptidyl carboxypeptidase pathway, while cathepsin B displays a preference for the dipeptidyl pathway. The preference for one or the other pathway is about the same for both enzymes, i.e., approximately 2 orders of magnitude, a result supported by molecular modeling of enzyme-substrate complexes. Cleavage of a C-terminal dipeptide of a substrate by cathepsin X can become more important under conditions that preclude efficient monopeptidyl carboxypeptidase activity, e.g., nonoptimal interactions in subsites S(2)-S(1). These results confirm that cathepsin X is designed to function as a monopeptidyl carboxypeptidase. Contrary to a recent report [Klemencic, I., et al. (2000) Eur. J. Biochem. 267, 5404-5412], it is shown that cathepsins X and B do not share similar activity profiles, and that reagents are available to clearly distinguish the two enzymes. In particular, CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. The insights obtained from this and previous studies have been used to produce an inhibitor designed to exploit the unique structural features responsible for the carboxypeptidase activity of cathepsin X. Although of moderate potency, this E-64 derivative is the first reported example of a cathepsin X-specific inhibitor.
The Slow-Binding Inhibition of Cathepsin K by Its Propeptide
Biochemical and Biophysical Research Communications, 2000
A peptide corresponding to the full-length proregion (amino acids 16 -114) of human cathepsin K was expressed and purified from Escherichia coli. This recombinant propeptide was investigated for its ability to inhibit the activity of three cysteine proteinases: cathepsins K, L, and B. Kinetic studies showed the propeptide to be a potent slow-binding inhibitor of its parent enzyme with a K i ؍ 2.61 nM at pH 6. This inhibition was pH-dependent, with a decrease in pH from 6 to 4 leading to a concomitant increase in K i to 147 nM. The propeptide also inhibited cathepsin L with a K i ؍ 26.1 nM at pH 6, but showed little inhibition of cathepsin B at concentrations up to 400 nM.
Inhibition of a Cathepsin L-Like Cysteine Protease by a Chimeric Propeptide-Derived Inhibitor †
Biochemistry, 2005
Like other papain-related cathepsins, congopain from Trypanosoma congolense is synthesized as a zymogen. We have previously identified a proregion-derived peptide (Pcp27), acting as a weak and reversible inhibitor of congopain. Pcp27 contains a 5-mer YHNGA motif, which is essential for selectivity in the inhibition of its mature form []. In the work presented here, a homology model of procongopain was generated and subsequently used to model a chimeric 50mer peptide (called H3-Pcp27) corresponding to the covalent linkage of an unrelated peptide (H3 helix from Antennapedia) to Pcp27. Molecular simulations suggested that H3-Pcp27 (pI ) 9.99) maintains an N-terminal helical conformation, and establishes more complementary electrostatic interactions (E coul ) -25.77 kcal/mol) than 16N-Pcp27, the 34-mer Pcp27 sequence plus the 16 native residues upstream from the proregion (E coul ) 0.20 kcal/mol), with the acid catalytic domain (pI ) 5.2) of the mature enzyme. In silico results correlated with the significant improvement of congopain inhibition by H3-Pcp27 (K i ) 24 nM), compared to 16N-Pcp27 (K i ) 1 µM). In addition, virtual alanine scanning of H3 and 16N identified the residues contributing most to binding affinity. Both peptides did not inhibit human cathepsins B and L. In conclusion, these data support the notion that the positively charged H3 helix favors binding, without modifying the selectivity of Pcp27 for congopain.
European Journal of Biochemistry, 2003
Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of < 33 kDa and pI 5.1±5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (K i 1.7±15.0 nm), but poorly or not at all by stefin B (K i . 250 nm) and l-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1 H position, although the enzyme cleaved all P1 H residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (k cat /K m < 5.0 Â 10 3 m 21´s21 ) were degraded < 25-fold less efficiently than the carboxypeptidase substrates (k cat /K m < 120.0 Â 10 3 m 21´s21 ).
European Journal of Biochemistry, 2000
Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of < 33 kDa and pI 5.1±5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (K i 1.7±15.0 nm), but poorly or not at all by stefin B (K i . 250 nm) and l-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1 H position, although the enzyme cleaved all P1 H residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (k cat /K m < 5.0 Â 10 3 m 21´s21 ) were degraded < 25-fold less efficiently than the carboxypeptidase substrates (k cat /K m < 120.0 Â 10 3 m 21´s21 ).
European Journal of Biochemistry, 2000
Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.