A study of the complex of proteolytic enzymes and their protein inhibitors in embryogenesis of the silkworm

We studied changes in the activities of serin, thiol, and aspartyl proteinases and their protein inhibitors during embryogenesis of the silkworm. The dynamics of activities of the protein inhibitors and specific proteinases were interrelated, thus providing for coordination and fine regulation of functioning of the proteolytic complex of enzymes during embryogenesis. Possible functions of peptidohydrolases and their protein inhibitors in the silkworm are discussed.     

An analysis of intracellular proteinases from antibiotic-producing cells of Penicillium urticae

Two distinct patterns of activity obtained with the substrates azocasein and azocollagen suggested that Penicillium urticae produces at least two intracellular proteinases during its antibiotic-production phase. Cell extracts fractionated using high resolution gel filtration actually separated three major activities. These three pooled fractions contained predominantly cysteine-type proteinases, as indicated by their sensitivities to inhibitors and by the enhancement of their activities with dithiothreitol and ethylenediamine-tetracetic acid. One of these fractions also appeared to contain significant levels of serine-type proteinases. The three pools could be differentiated from one another by changes in their substrate specificities over a range of pH values, and by their stabilities during storage and at elevated temperatures. Further purification by non-denaturing polyacrylamide gel electrophoresis, revealed that two of the fractions each contained five apparently different activities while in the third pooled fraction, thirteen individual activities were detected. The range of properties displayed by these proteinases is consistent with their probable involvement in general protein degradation, a crucial process which during starvation sustains the supply of substrate necessary for secondary enzyme synthesis as well as contributing to the short lifetime of these same secondary enzymes.     

Proteomic analysis of larval integument, trachea and adult scale from the silkworm, Bombyx mori

Multidimensional LC-tandem MS was used to investigate the protein compositions of three tissues of silkworm, Bombyx mori. A total of 162, 259, and 175 peptides from silkworm larval integument and trachea, and adult scale obtained by database search were matched to 48, 51, and 40 proteins, respectively. Forty-one cuticular proteins were identified from three tissues and covered all five cuticular protein families of silkworm. In the adult scale, all seven cuticular proteins were identified for the first time in the final pellet after SDS extraction. The majority of cuticular proteins were found in each tissue differentially, suggesting that tissue-specific cuticular proteins were involved in the building of the specialized tissues. Seventy-three non-cuticular proteins were also identified in this analysis mainly including muscular proteins, proteinases, inhibitors, transport proteins, and redox-related proteins.     

Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens

Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.     

Effect of low-temperature hardening on activities of proteolytic enzymes and their inhibitors in the leaves of wheat and cucumber seedlings

On seedlings of winter wheat (Triticum aestivum L.) and cucumber (Cucumis sativus L.), the dynamics of cysteine and serine trypsin-like proteinases and also trypsin inhibitors at cold hardening (5°C for wheat and 10°C for cucumber) was studied. Activation of proteinases and inhibitors coincided in time or preceded an increased tolerance in wheat and cucumber seedlings in the early period of their hardening. After attaining the highest wheat tolerance, activity amidases reduced, whereas the increased activity levels of cysteine proteinases and trypsin inhibitors was maintained during the entire period of hardening. In cucumber, in these period activities of amidases and trypsin inhibitors reduced, whereas the activity of cysteine proteinases was maintained at the level close to the initial one. It is suggested that cysteine proteinases, amidases, and trypsin inhibitors are involved in plant adaptation to cold.     

Proteolytic enzymes as markers of productivity and heterosis of silkworm

A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating a principal possibility of predicting the degree of heterosis.     

金鱼不同组织器官及胚胎发育不同时期蛋白水解酶的种类和活性变化

采用复性电泳方法研究了金鱼组织器官蛋白水解酶及个体发生过程中蛋白水解酶的种类和活性变化,主要结果表明：⑴金鱼各组织器官蛋白水解酶种类差异不大,大多数组织器官都具有１１３、６９、２０、１６ｋＤ四条带,但不同组织器官常具有其特异性蛋白水解酶;肠道蛋白水解酶种类最多、活性最强。⑵蛋白水解酶的活性受ｐＨ值影响和制约,大多数组织器官蛋白水解酶活性最适ｐＨ值为８．５。⑶在金鱼胚胎发育早期（从卵裂到心跳期）多数     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Proteinase,Lipase and Amylase Activities in the Small Intestine of Pigs Suffering from Colienterotoxaemia

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test. Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents. In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers. The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

Effect of macrophage differentiation and exposure to mildly oxidized LDL on the proteolytic repertoire of THP-1 monocytes

Lipid-laden monocyte/macrophages in atherosclerotic plaques can produce a range of proteinases capable of degrading components of the plaque extracellular matrix, an event that may weaken plaques, rendering them vulnerable to rupture. The effects of differentiation from monocytes to macrophages and exposure to mildly oxidized LDL (Ox-LDL) on the expression of a range of proteinases and their inhibitors were assessed in the human THP-1 cell line. Of 56 proteinases/inhibitors investigated, 17 were upregulated during macrophage differentiation, including several matrix metalloproteinases (MMPs) and cathepsins along with their native inhibitors. Similarly, expression of matrix-degrading proteinases was also increased during differentiation of human primary macrophages. In conjunction, the proteolytic capacity of the cells increased, as assessed by substrate zymography. Subsequent exposure of differentiated THP-1 cells to mildly Ox-LDL increased the expression of a control gene (adipocyte lipid binding protein) and increased the activity of nuclear factor-kappaB and activator protein-1 in serum-free conditions but did not significantly affect the expression of any of the proteinases or inhibitors investigated. These results indicate that in this model macrophage differentiation, rather than exposure to Ox-LDL, has a more important effect on the expression of genes involved in extracellular matrix remodeling.     

家蚕胚胎期重力相关基因的抑制消减杂交分析

目的：筛选家蚕胚胎期重力相关基因。方法：对模拟失重与正常重力条件下的家蚕胚胎cDNA进行抑制消减杂交（Suppression subtractive hybridization,SSH）,并对模拟失重过程中家蚕胚胎期表达发生变化的基因进行克隆、测序及同源性分析。结果：获得了34个与重力有关的序列标签。在模拟失重条件下有16个基因表达上调,其中15个为未知基因,1个为已知基因,其作用是维持mRNA的稳定性。在模拟失重条件下有18个基因表达下调,其中4个为未知基因,6个为蛋白合成相关基因,3个为基因组contig基因,5个为家蚕est库中功能未知基因。结论：模拟失重环境影响了家蚕胚胎发育期与mRNA稳定性和蛋白质合成相关基因的表达。     

Proteolytic enzymes as markers of productivity and heterosis of silkworm

A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating the possibility of predicting the degree of heterosis.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 99–106.Original Russian Text Copyright © 2005 by Krylova, Yarygin, Filippovich.     

Serine protease P-IIc is responsible for the digestion of yolk proteins at the late stage of silkworm embryogenesis

In silkworms, yolk proteins comprise vitellin, egg-specific protein and 30K proteins, which are sequentially degraded by endogenous proteases strictly regulated during embryogenesis. Although the process has been extensively investigated, there is still a gap in the knowledge about the degradation of silkworm yolk proteins on the last two days of embryonic development. In the present study, we isolated and purified a gut serine protease P-IIc, which demonstrated optimal activity at 25 °C and pH 11. Semi-quantitative RT-PCR combined with western blotting showed that P-IIc was actively expressed and significantly accumulated in the gut on the last two days of embryogenesis. When natural yolk proteins were incubated with P-IIc in vitro, vitellin and ESP were selectively degraded. P-IIc also demonstrated activity towards 30K proteins as evidenced by rapid and complete digestion of BmLP1 and partial digestion of BmLP2 and BmLP3. Furthermore, RNAi knockdown of P-IIc in silkworm embryos significantly reduced the degradation rate of residual yolk proteins on embryonic day 10. Taken together, our results indicate that P-IIc represents an embryonic gut protease with a relatively broad substrate specificity, which plays an important role in the degradation of yolk proteins at the late stage of silkworm embryogenesis.     

The cystatins: protein inhibitors of cysteine proteinases.

The last decade has witnessed enormous progress of protein inhibitors of cysteine proteinases concerning their structures, functions and evolutionary relationships. Although they differ in their molecular properties and biological distribution, they are structurally related proteins. All three inhibitory families, the stefins, the cystatins and the kininogens, are members of the same superfamily. Recently determined crystal structures of chicken cystatin and human stefin B established a new mechanism of interaction between cysteine proteinases and their inhibitors which is fundamentally different from the standard mechanism for serine proteinases and their inhibitors.     

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF-MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.     

Study of intracellular localization of the proteolytic enzyme complex and its protein inhibitors in bombyx grain

Intracellular localization of serine, cysteine and aspartate proteases, as well as their protein inhibitors, in bombyx grain in the postdiapause period of embryogenesis has been studied. Proteolytic activity of aspartate and cysteine proteases was found in lysosomal, mitochondrial, and nuclear fractions of grains. Serine protease activity was not observed in subcellular fractions of grains of the fourth day of postdiapause development. It has been shown that activities of protein inhibitors and certain peptide hydrolases in subcellular fractions provide consistent functioning and fine regulation of the proteolytic enzyme complex.     

A sensitive polyacrylamide disc gel method for detection of proteinases

To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.     

Up-regulation of multiple serine proteinases during earthworm tail regeneration

Summary

Previous studies have shown that spatiotemporal regulation of extracellular matrix (ECM) by proteinases is implicated in the initial step of regeneration. In amphibian regeneration, the up-regulation of proteinases such as metalloproteinases (MMPs) and cathepsin D, and proteinase-related proteins such as proteinase tissue inhibitors and activators has been demonstrated. Since the earthworm could provide a unique and valuable model to investigate the mechanism of regeneration, we studied the developmental change in proteinase expression during earthworm tail regeneration. Zymographic analysis revealed that proteinase activities began to increase within 1 h after amputation and reached a maximum at 7 days post-amputation. This peak in activity was approximately 22-fold greater than the unamputated controls. Thereafter, the proteinase activities tended to decrease followed by another peak at 30 days before returning to control levels. At least four types of proteinase were distinguishable at 7 and 30 days post-amputation, with molecular weights of 25, 28, 38, and 44 kDa, respectively. All proteinase activities were strongly inhibited by addition of phenylmethylsulfonyl fluoride (PMSF) and aprotinin, specific inhibitors for serine proteinase. Pepstatin A, E-64, iodoacetamide and a metal ion-free medium were not effective inhibitors, indicating that proteinases expressed during earthworm tail regeneration would be serine proteinases. In addition, we were able to detect two types of plasminogen activator (PA) with molecular weights of 40 and 47 kDa, respectively. PA activities were predominantly expressed at 1, 5, and 25 days post-amputation, which preceded two peaks of serine proteinase activities appearing at approximately 7 and 30 days after amputation, respectively. This fact supports the view that serine proteinases expressed in respond to tail amputation may be plasmin-like proteinases activated by PA.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.     

Mutants of plasminogen activator inhibitor-1 designed to inhibit neutrophil elastase and cathepsin G are more effective in vivo than their endogenous inhibitors

Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.