Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane-permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane-permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of β-glucuronised, and N-acetyle-β-glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does-dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.     

Residue-residue contact substitution probabilities derived from aligned three-dimensional structures and the identification of common folds.

We report the derivation of scores that are based on the analysis of residue-residue contact matrices from 443 3-dimensional structures aligned structurally as 96 families, which can be used to evaluate sequence-structure matches. Residue-residue contacts and the more than 3 x 10(6) amino acid substitutions that take place between pairs of these contacts at aligned positions within each family of structures have been tabulated and segregated according to the solvent accessibility of the residues involved. Contact maps within a family of structures are shown to be highly conserved (approximately 75%) even when the sequence identity is approaching 10%. In a comparison involving a globin structure and the search of a sequence databank (> 21,000 sequences), the contact probability scores are shown to provide a very powerful secondary screen for the top scoring sequence-structure matches, where between 69% and 84% of the unrelated matches are eliminated. The search of an aligned set of 2 globins against a sequence databank and the subsequent residue contact-based evaluation of matches locates all 618 globin sequences before the first non-globin match. From a single bacterial serine proteinase structure, the structural template approach coupled with residue-residue contact substitution data lead to the detection of the mammalian serine proteinase family among the top matches in the search of a sequence databank.     

Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by "4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca²⁺-dependent while lysis is delayed in the absence of exogenous Ca²⁺. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.Abbreviations TPCK Tos-Phe-CH₂Cl,1-chloro-4 phenyl-3-L-tosylamidobutan-2-one - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - FCS Fetal Calf Serum     

Partial characterization of digestive tract proteinases from western corn rootworm larvae,Diabrotica virgifera

We have partially characterized the proteolytic activity within luminal contents of the digestive tracts of larvae of the western corn rootworm, Diabrotic virgifera LeConte. At least 15 proteinases were detected based on chromatographic behavior on ion exchange high-performance liquid chromatography and their mobility on sodium dodecyl sulfate gels containing gelatin. Inhibitors of proteolytic activity indicated that these enzymes are primarily sulfhydryl proteinases. Native polycrylamide gel electrophoresis revealed that a single proteinaceous inhibitor, egg white cystatin, was capable of abolishing a substantial part of the proteolytic acitivity. Our data suggest, accordingly, that gene transfer experiments utilizing the cystatin gene may generate lines of maize which have increased resistance to western corn rootworm larvae. © 1992 Wiley-Liss, Inc.     

Long-term effects of soybean protease inhibitors on digestive enzymes, survival and learning abilities of honeybees

We have investigated the effects of long-term ingestion of two serine proteinase inhibitors (PIs), the Kunitz Soybean trypsin inhibitor (SBTI) and the Bowman-Birk inhibitor (BBI) on survival, learning abilities involved in the foraging behaviour, and digestive physiology of the honeybee (Apis mellifera L., Hymenoptera). A threshold-dose was established, above which adverse effects of long-term ingestion of the PIs tested are to be expected. The experiments reported herein could be extended to other PIs or gene products used to confer insect resistance, and be part of a general procedure used to assess the innocuousness of transgenic melliferous plants to honeybees.     

点状产气单胞菌脯氨酰内肽酶基因克隆与序列测定

点状产气单胞菌点状亚种（Ａｅｒｏｍｏｎａｓ ｐｕｎｃａｔａ ｓｕｂｓｐ．ｐｕｎｃｔａｔａ）具有脯氨酰内肽酶（ｐｒｏｌｙｌ ｅｎｄｏｐｅｐｔｉｄａｓｅ ＰＥＰ）活性。将其染色体ＤＮＡ有Ｅｃｏ ＲⅠ部分酶切后回收８－１６ｋｂ的ＤＮＡ片段,与ＥｃｏＲⅠ消化载体ｐＵＣ１８连接后转化Ｅ．ｃｏｉｌ ＤＨ５α,用该酶的专一性底物Ｂｅｎｚｙｌｏｘｙｃａｒｂｏｎｙｌ－Ｇｌｙ－β－ｎａｐｈｔｈｙｌａｍｉｄｅ从质粒库中     

Mapping the active site of factor Xa by selective inhibitors: An NMR and MD study

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH₂ and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH₂, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site. Proteins 30:264–279, 1998. © 1998 Wiley-Liss, Inc.     

Molecular mechanisms of antithrombin–heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors

Serpin family protein proteinase inhibitors regulate the activity of serine and cysteine proteinases by a novel conformational trapping mechanism that may itself be regulated by cofactors to provide a finely-tuned time and location-dependent control of proteinase activity. The serpin, antithrombin, together with its cofactors, heparin and heparan sulfate, perform a critical anticoagulant function by preventing the activation of blood clotting proteinases except when needed at the site of a vascular injury. Here, we review the detailed molecular understanding of this regulatory mechanism that has emerged from numerous X-ray crystal structures of antithrombin and its complexes with heparin and target proteinases together with mutagenesis and functional studies of heparin–antithrombin–proteinase interactions in solution. Like other serpins, antithrombin achieves specificity for its target blood clotting proteinases by presenting recognition determinants in an exposed reactive center loop as well as in exosites outside the loop. Antithrombin reactivity is repressed in the absence of its activator because of unfavorable interactions that diminish the favorable RCL and exosite interactions with proteinases. Binding of a specific heparin or heparan sulfate pentasaccharide to antithrombin induces allosteric activating changes that mitigate the unfavorable interactions and promote template bridging of the serpin and proteinase. Antithrombin has thus evolved a sophisticated means of regulating the activity of blood clotting proteinases in a time and location-dependent manner that exploits the multiple conformational states of the serpin and their differential stabilization by glycosaminoglycan cofactors.     

Pig IαI appears unmodified in plasma in case of endotoxin-induced disseminated intravascular coagulation

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-α-inhibitor (IαI) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a procine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine IαI in plasmas collected from pigs after endotoxin infusion. To explain these data. IαI was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, procine cells were unable to proteolyze IαI. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.