Biomedical subjects

M A Juliano

Publications and source records attributed to M A Juliano.

At least 19 recordsLinked to original sources

PubMed2006

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates.

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.

Amino Acids, Basic↗

PubMed2006

The C-terminus of murine S100A9 protein inhibits hyperalgesia induced by the agonist peptide of protease-activated receptor 2 (PAR2).

BACKGROUND AND PURPOSE: S100A9 protein induces anti-nociception in rodents, in different experimental models of inflammatory pain. Herein, we investigated the effects of a fragment of the C-terminus of S100A9 (mS100A9p), on the hyperalgesia induced by serine proteases, through the activation of protease-activated receptor-2 (PAR2). EXPERIMENTAL APPROACH: Mechanical and thermal hyperalgesia induced by PAR2 agonists (SLIGRL-NH2 and trypsin) was measured in rats submitted to the paw pressure or plantar tests, and Egr-1 expression was determined by immunohistochemistry in rat spinal cord dorsal horn. Calcium flux in human embryonic kidney cells (HEK), which naturally express PAR2, in Kirsten virus-transformed kidney cells, transfected (KNRK-PAR2) or not (KNRK) with PAR2, and in mouse dorsal root ganglia neurons (DRG) was measured by fluorimetric methods. KEY RESULTS: mS100A9p inhibited mechanical hyperalgesia induced by trypsin, without modifying its enzymatic activity. Mechanical and thermal hyperalgesia induced by SLIGRL-NH2 were inhibited by mS100A9p. SLIGRL-NH2 enhanced Egr-1 expression, a marker of nociceptor activation, and this effect was inhibited by concomitant treatment with mS100A9p. mS100A9p inhibited calcium mobilization in DRG neurons in response to the PAR2 agonists trypsin and SLIGRL-NH2, but also in response to capsaicin and bradykinin, suggesting a direct effect of mS100A9 on sensory neurons. No effect on the calcium flux induced by trypsin or SLIGRL in HEK cells or KNRK-PAR2 cells was observed. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that mS100A9p interferes with mechanisms involved in nociception and hyperalgesia and modulates, possibly directly on sensory neurons, the PAR2-induced nociceptive signal.

Analgesics↗

PubMed2005

Substrate specificity of insect trypsins and the role of their subsites in catalysis.

Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1', P2', P2, and P3. The binding energy (deltaG(s), calculated from Km values) and the activation energy (deltaG(T)(double dagger), determined from kcat/Km values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P1 at least 3 times more efficiently than peptides with Lys at P1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1', and S2' significantly bind the substrate ground state, whereas in the transition state only S1' and S2' do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties.

Animals↗

PubMed2005

A continuous fluorescent assay for the determination of plasma and tissue angiotensin I-converting enzyme activity.

A continuous assay using internally quenched fluorescent peptides with the general sequence Abz-peptidyl-(Dnp)P-OH (Abz = ortho-aminobenzoic acid; Dnp = 2,4-dinitrophenyl) was optimized for the measurement of angiotensin I-converting enzyme (ACE) in human plasma and rat tissues. Abz-FRK(Dnp)P-OH, which was cleaved at the Arg-Lys bond by ACE, was used for the enzyme evaluation in human plasma. Enzymatic activity was monitored by continuous recording of the fluorescence (lambda ex = 320 nm and lambda em = 420 nm) at 37 degrees C, in 0.1 M Tris-HCl buffer, pH 7.0, with 50 mM NaCl and 10 microM ZnCl2. The assays can be performed directly in the cuvette of the fluorimeter and the hydrolysis followed for 5 to 10 min. ACE measurements in the plasma of 80 healthy patients with Hip-His-Leu and with Abz-FRK(Dnp)P-OH correlated closely (r = 0.90, P < 0.001). The specificity of the assay was demonstrated by the complete inhibition of hydrolysis by 0.5 microM lisinopril or captopril. Abz-FRK(Dnp)P-OH cleavage by ACE was monitored in rat lung, kidney, heart, and liver homogenates in the presence of a cocktail of inhibitors containing trans-epoxy-succinyl-L-leucylamido-(4-guanido)-butene, pepstatin, phenyl-methylsulfonyl fluoride, N-tosyl-L-phenylalanyl-chloromethyl ketone, and N-tosyl-lysyl-chloromethyl ketone to prevent undesirable hydrolysis. ACE activity in lung, heart and kidney homogenates, but not in liver homogenates, was completely abolished by 0.5 microM lisinopril or captopril. The advantages of the method are the procedural simplicity and the high sensitivity providing a rapid assay for ACE determinations.

Animals↗

PubMed2005

The C-terminus of murine S100A9 inhibits spreading and phagocytic activity of adherent peritoneal cells.

OBJECTIVE AND DESIGN: In the present study, the effect of a synthetic peptide (H(92)-G(102)) identical to the C-terminus of murine S100A9 (mS100A9p) was investigated on adherent peritoneal cell function. MATERIALS AND METHODS: For in vitro assays, peritoneal cells were obtained from the abdominal cavity of mice and incubated, with the different concentrations of mS100A9p, for 1 h, and then their spreading and phagocytosis activities were evaluated. For ex-vivo assays, cells obtained from animals treated for 1 h with the peptide were submitted to the mannose-receptor phagocytosis assay. Shorter homologue peptides to the C-terminus of mS100A9p were also evaluated on in vitro phagocytosis assays of Candida albicans particles. RESULTS: mS100A9p reduced both the spreading index and phagocytic activity, in vitro and ex-vivo, independent of the receptor evaluated. The homologue peptide corresponding to the H(92)-E(97) region of mS100A9p, the zinc-binding motif, was responsible for such an effect. CONCLUSION: These results suggest a modulator effect of the C-terminus of S100A9 protein on the function of adherent peritoneal cells.

Amino Acid Motifs↗

PubMed2004

Coevolution of insect trypsins and inhibitors.

Many plant proteinase inhibitors have lysine at the P1 position, presumably to avoid hydrolysis by insect trypsins. Lepidopteran trypsins appear to have adapted to resist proteinase inhibitors through increased inhibitor hydrolysis and decreased binding to inhibitor hydrophilic reactive sites. Lepidopteran digestive trypsins prefer lysine at the P1 position and have substrate binding subsites more hydrophobic than trypsins from insects in other orders. All available sequences of sensitive and inhibitor-insensitive insect trypsins were aligned with porcine trypsin, for which interactions with Kunitz and Bowman-Birk inhibitor are known. After discounting conserved positions and positions not typical of sensitive or insensitive trypsins, the following residues were considered important to insect trypsin-PI interactions (chymotrypsin numbering): 60, 94, 97, 98, 99, 188, 190, 213, 215, 217, 219, 228. These residues support the Neighbor Joining analysis tree branches separating sensitive and insensitive trypsin sequences. Primary sequences interacting with PIs are around the active site, with some forming part of the S1 (188, 217, 219 and 228) or S4 (99, 215) pockets.

Amino Acid Sequence↗

PubMed2004

Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine.

In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].

Amino Acid Sequence↗

PubMed2003

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells.

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Animals↗

PubMed2003

Mapping of human plasma kallikrein active site by design of peptides based on modifications of a Kazal-type inhibitor reactive site.

Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P'1, P'3 and P'4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P'4 position is important for enzyme action.

Amino Acid Sequence↗

PubMed2003

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Amino Acid Sequence↗

PubMed2002

Subsites of trypsin active site favor catalysis or substrate binding.

Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T), determined from k(cat)/K(m) values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(s) for oligopeptides with 15 amino acid replacements at each of the positions P(1)', P(1), and P(2) were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(s). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis.

Amino Acids↗

PubMed2001

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation.

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.

Animals↗

PubMed2001

Characterization of two cysteine proteinases secreted by Fasciola hepatica and demonstration of their kininogenase activity.

We have isolated and purified two cysteine proteinases of molecular weights 25 and 26 kDa, secreted by Fasciola hepatica adult worm. Their 15 N-terminal residues were found to be identical to those of earlier described cathepsin L-like enzymes, isolated from the same source, reported as CL1 and CL2. Radioimmunoassay experiments have shown that these CL1- (25 kDa) and CL2-like (26 kDa) cysteine proteinases mediated kinin release from high molecular weight kininogen (HMWK). Lys-bradykinin (KRPPGFSPFR) was characterized as the kinin released from a synthetic fragment of HMWK from Leu373 to Ile393 (Abz-LGMISLMKRPPGFSPFRSSRI-NH2) labeled with the fluorescent group Abz (ortho-aminobenzoic acid). We examined the activity of CL1- and CL2-like on internally quenched fluorescent peptides containing HMWK sequences, in which Met379-Lys380 or Arg389-Ser390 bonds were present in the middle of the molecules. These peptides were flanked by the fluorescent donor-acceptor pair Abz and EDDnp (N-[2,4-dinitrophenyl] ethylenediamine). Peptidyl-methylcoumarin amides (MCA) were used to study the substrate specificity requirements. The enzymes presented significantly lower Km values at pH 8.0. The inverse was observed with the kcat values, which were higher at pH 5.0.

Amides↗

PubMed2001

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides.

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.

Cathepsin B↗

PubMed2001

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11).

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.

Alanine↗

PubMed2001

Synthesis and hydrolysis by cathepsin B of fluorogenic substrates with the general structure benzoyl-X-ARG-MCA containing non-natural basic amino acids at position X.

We synthesized one series of fluorogenic substrates for cathepsin B derived from the peptide Bz-F-R-MCA (Bz=benzoyl, MCA=7-methyl-coumarin amide) substituting Phe at the P(2) position by non-natural basic amino acids that combine a positively charged group with aromatic or aliphatic radicals at the same side chain, namely, 4-aminomethyl-phenylalanine, 4-guanidine-phenylalanine, 4-aminomethyl-N-isopropyl-phenylalanine, 3-pyridyl-alanine, 4-piperidinyl-alanine, 4-aminomethyl-cyclohexyl-alanine, 4-aminocyclohexyl-alanine, and N(im)-dimethyl-histidine. Bz-F-R-MCA was the best substrate for cathepsin B but also hydrolyzed Bz-R-R-MCA with lower efficiency, since the protease accepts Arg at S(2) due to the presence of Glu(245) at the bottom of this subsite. The presence of the basic non-natural amino acids at the P(2) position of the substrate partially restored the catalytic efficiency of cathepsin B. All the kinetic parameters for hydrolysis of the peptides described in this paper are in accordance with the structures of the S(2) pocket previously described. In addition, the substrate with 4-aminocyclohexyl-alanine presented the highest affinity to cathepsin B although the peptide was obtained from a mixture of cis/trans isomers of the amino acid and we were not able to separate them. For comparison all the obtained substrates were assayed with cathepsin L and papain.

Amino Acids, Diamino↗

PubMed2001

Human tissue kallikrein S1 subsite recognition of non-natural basic amino acids.

We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.

Amino Acid Sequence↗

PubMed2001

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana.

We have identified peptides that are relatively resistant to hydrolysis by a recombinant cysteine proteinase, CPB2.8DeltaCTE, of Leishmania mexicana, and yet exhibit inhibition constant (K(i)) values in the nanomolar range. Common to these peptides is a basic-hydrophobic-hydrophobic motif in the P3-P1 sites, which is also present in the pro-region of the enzyme. A nine-amino acid stretch, FAARYLNGA, which has good homology to the pro-region of mammalian cathepsin L was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme. This peptide is not hydrolyzed by CPB2.8DeltaCTE and inhibited it with a K(i) of 4 microM. Extension of this sequence at both the N- and C-termini and the introduction of ortho-aminobenzoic acid at the N-terminal site reduced the K(i) value to 30 nM. The best substrate for CPB2.8DeltaCTE was also well hydrolyzed by cathepsin L, however the best inhibitor of the parasite enzyme inhibit poorly cathepsin L, with K(i) value two order of magnitude higher than against the parasite enzyme. These promising data provide insights into the peculiar specificity of the parasite enzyme and will aid the design of antiparasitic drugs directed against the leishmanial enzyme.

Amino Acid Sequence↗