Propeptide-Mediated Specific Inhibition of a Recombinant Serine Protease from Indian Malaria Vector, Anopheles culicifacies

Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles c ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its three deletion mutants and leupeptin (a general serine protease inhibitor) were tested in vitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.     

Characterization of Insect Proteinases and Their Inhibition by Finger and Little Millet Inhibitors

Empirical approach was adopted to examine the interaction of a little and finger millet inhibitors with digestive proteinases ofdifferent field and storage pests. Accordingly, gut proteinases of four storage and phytophagous pests were characterized andtheir inhibition by finger millet (Eluesine coracana Gaertneri) and little millet (Panicum sumatrense Roth) inhibitors has beeninvestigated. The Callosobruchus sp showed acid proteinase with pH optimum of 3.5 and Sitophilus oryzae showed pHoptimum of 4.5 apart from their alkaline proteinases with pH 8.5. The proteinases of Tribolium castaneum had wider pHoptimum from 5.5 to 8.5. However, all lepidopteran insect proteinases had pH optima ranging from 8.5 to 10.5. The optimumtemperature was found to be 30 to 40 °C. The inhibitory activities of little and finger millet inhibitors towards the insectproteinases are very low except notable level inhibition of proteinases of some insects. The gut proteinase zymogram ofdifferent insects revealed 2 – 6 isozymes and the inhibitors moderately inhibited all the isozymes of insects tested.     

Purification of Yeast Proteinases

The presence of three types of proteinase A, B and C in the autolysate of baker’s yeast was demonstrated by the chromatography on DEAE-Sephadex A-50, and proteinase A and C were isolated and purified by a relatively simple procedure, which was mainly conducted by repeating the chromatography and alcohol fractionation. The final preparation of proteinase C was found to be homogeneous by various physical criteria and crystallized from alcohol solution. On the other hand, although the preparation of proteinase A also showed homogeneous in chromatographic and ultracentrifugal analyses, the result of electrophoresis disclosed the heterogeneity of the preparation. As the results of the chemical and physicochemical analyses, both enzymes showed large contents of carbohydrate, higher molecular weights and acidic isoelectric points, which seemed to be characteristic to the present proteinases. The properties of three types of proteinase from yeast are also discussed.     

Substrate Specificity and Salt Inhibition of Five Proteinases Isolated from the Pyloric Caeca and Stomach of Sardine

To elucidate the mechanism of hydrolysis of fish muscle proteins by fish proteinases in fish sauce production, each pure preparation of three alkaline proteinases and two acid proteinases from sardine was tested for its ability to hydrolyze various proteins and its stability in the presence of 0 to 25% of NaCl. Each of the alkaline proteinases hydrolyzed casein more rapidly than other proteins. A major alkaline proteinase (III) hydrolyzed sarcoplasmic protein from sardine 5-times faster than other alkaline proteinases. Each of two acid proteinases hydrolyzed hemoglobin and myoglobin more rapidly than the other proteins. After preincubation with 25% NaCl, an alkaline proteinase (III) and an acid proteinase (II) were stable although the other proteinases became unstable. The two proteinases, alkaline proteinase III and acid proteinase II, were also stable for three months after the beginning of fish sauce production. The proteolytic activity of each of alkaline and the acid proteinases was strongly inhibited by more than 15% NaCl; however, minimum inhibition was observed when sardine muscle proteins were used as the substrate.     

Proteinases in fungal morphogenesis

Fungal morphogenesis is a regulated series of events, leading to changes from one state to another, in which proteolysis could be regarded as one of the controlling functions. Proteinases are essential for the supply of amino acids, selective inactivation of specific growth phase proteins not required during development and for the activation and modification of the enzymes involved in cell wall synthesis. A critical evaluation of the role of proteinases as a biochemical correlate in fungal morphogenesis is discussed.     

基质金属蛋白酶ADAMTS的研究进展

ADAMTS是含有TSP基序的分泌性多结构域蛋白水解酶。ADAMTS基因的突变或过表达与多种生理学和病理学过程相关。ADAMTS基因的过表达促进了细胞外基质成分的降解,加速了关节炎和动脉粥样硬化的疾病进程。ADAMTS基因的突变则与肿瘤的生长和侵袭以及遗传性发育紊乱等密切相关。该文将结合作者的研究工作,重点对ADAMTS-7的当前研究概况进行综述,讨论其结构、功能、调节及其在相关炎性疾病中的作用等。     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

Cationic Inhibitors of Serine Proteinases from Buckwheat Seeds: Study of Their Interaction with Exogenous Proteinases

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.     

Binding Specificity of the Porphyromonas gingivalis Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in Porphyromonas gingivalis is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant P. gingivalis HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to Escherichia coli cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 x 10(-5) M. Recombinant E. coli cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998     

Studies on Proteinases from Gliocladium roseum

The alkaline proteinases of Gliocladium roseum (Link) Bainier were purified in crystalline forms by procedures of alcoholic precipitation, fuller’s earth- and acrinol-treatment, and isolated in two types. (Proteinases I and II). Both of these proteinases were homogeneous on zone electrophoresis with polyacrylamide gel (Gyanogum 41), and had the optimal pH values of 11 (Proteinase I) and 10 (Proteinase II), and the optimal temperature of 45°C.

The enzymatic reaction of proteinase I was remarkably promoted by Fe⁺⁺ and Co⁺⁺, and that of proteinase II was promoted by Fe⁺⁺, Go⁺⁺ and Ca⁺⁺, and both proteinases were protected from heat-inactivation by Ca⁺⁺ Proteinase II was activated remarkably by Cl^? under the existence of Fe⁺⁺, but proteinase I was unaffected by the anion.

The order of strength of proteolytic power of these proteinases and chymotrypsin on casein was as follows; proteinase I> proteinase II> chymotrypsin.     

Proteinases in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide, and we have little specific therapy to offer these patients. One potential strategy to limit loss of lung function in COPD would be to inhibit matrix-degrading proteinases. Several serine proteinases and matrix metalloproteinases are expressed in association with COPD in humans. Application of gene-targeted macrophage elastase and neutrophil elastase to a mouse model of cigarette-smoke-induced emphysema has uncovered roles for these proteinases in airspace enlargement, and has identified many interactions between these proteolytic systems.     

Cognate ligand domain mapping for enzymes

Here, we present an automatic assignment of potential cognate ligands to domains of enzymes in the CATH and SCOP protein domain classifications on the basis of structural data available in the wwPDB. This procedure involves two steps; firstly, we assign the binding of particular ligands to particular domains; secondly, we compare the chemical similarity of the PDB ligands to ligands in KEGG in order to assign cognate ligands. We find that use of the Enzyme Commission (EC) numbers is necessary to enable efficient and accurate cognate ligand assignment. The PROCOGNATE database currently has cognate ligand mapping for 3277 (4118) protein structures and 351 (302) superfamilies, as described by the CATH and (SCOP) databases, respectively. We find that just under half of all ligands are only and always bound by a single domain, with 16% bound by more than one domain and the remainder of the ligands showing a variety of binding modes. This finding has implications for domain recombination and the evolution of new protein functions. Domain architecture or context is also found to affect substrate specificity of particular domains, and we discuss example cases. The most popular PDB ligands are all found to be generic components of crystallisation buffers, highlighting the non-cognate ligand problem inherent in the PDB. In contrast, the most popular cognate ligands are all found to be universal cellular currencies of reducing power and energy such as NADH, FADH2 and ATP, respectively, reflecting the fact that the vast majority of enzymatic reactions utilise one of these popular co-factors. These ligands all share a common adenine ribonucleotide moiety, suggesting that many different domain superfamilies have converged to bind this chemical framework.     

Proteinases of potential objects of fish nutrition

The review analyzes data on activities and characteristics of proteinases of digestive system and of cathepsins of various tissues in potential preys of fish differing by the character of nutrition. There are presented data on multiple forms and molecular mass of proteinases, species-related differences, dependence of proteinase activities on the life cycle stages, type of nutrition, and biochemical food composition as well as on temperature and pH optima. Role of cathepsin in evolution of enzymatic systems providing degradation of food protein components in fish is discussed.     

Cognate restriction of transposition by piggyBac-like proteins

Mobile genetic elements have been harnessed for gene transfer for a wide variety of applications including generation of stable cell lines, recombinant protein production, creation of transgenic animals, and engineering cell and gene therapy products. The piggyBac transposon family includes transposase or transposase-like proteins from a variety of species including insect, bat and human. Recently, human piggyBac transposable element derived 5 (PGBD5) protein was reported to be able to transpose piggyBac transposons in human cells raising possible safety concerns for piggyBac-mediated gene transfer applications. We evaluated three piggyBac-like proteins across species including piggyBac (insect), piggyBat (bat) and PGBD5 (human) for their ability to mobilize piggyBac transposons in human cells. We observed a lack of cross-species transposition activity. piggyBac and piggyBat activity was restricted to their cognate transposons. PGBD5 was unable to mobilize piggyBac transposons based on excision, colony count and plasmid rescue analysis, and it was unable to bind piggyBac terminal repeats. Within the piggyBac family, we observed a lack of cross-species activity and found that PGBD5 was unable to bind, excise or integrate piggyBac transposons in human cells. Transposition activity appears restricted within species within the piggyBac family of mobile genetic elements.