Prognostic significance of lysosomal cysteine proteases in the estimation of the effectiveness of the antitumor therapy

The influence of the antitumor drugs, cyclophosphamide (CPA) and nitrosomethylurea (NMU) on the activity of lysosomal cysteine proteases cathepsin B and L in tumor tissue has been investigated using CPA-sensitive (LS) and CPA-resistant mouse lymphosarcomas (RLS). (These drugs exhibit high and low antitumor efficiency towards LS and RLS mouse lymphosarcomas, respectively). Regression or reduction in the growth rate of LS and RLS lymphosarcomas caused by CPA or NMU administration was accompanied by the increase in the activity of cysteine proteases cathepsin B and L in the tumor tissue. The increase of cathepsin B and L activity in tumor tissue correlated with the therapeutic effect of these drugs. Data obtained suggest that activity of cathepsin B and L in tumor tissue has a prognostic significance for the estimation of the effectiveness of antitumor therapy.     

Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases

So far nine human aspartate-specific cysteine proteases (ASCPs) have been identified and cloned in our lab and others. Their sequence and structural homology to the nematode Ced-3 implicated them in the cell death pathway of mammalian cells. Recent evidence suggests that ASCPs initiate apoptosis by acting at or near the cell death effector level. However, it is not clear whether the activity of one or several of these enzymes is necessary for execution of apoptosis. In addition, it is not yet clear how the proenzymes of ASCPs are activated or what triggers their activation. Execution of apoptosis in higher eukaryotes is apparently more complicated than in nematodes. It is most likely that in mammalian cells this process involves the coordinated action of multiple ASCPs and multiple redundant proteolytic pathways. J. Cell Biochem. 64:33–42. © 1997 Wiley-Liss, Inc.     

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K_I of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver–Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C′ in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.     

Estrogens, trypsin-like proteases and carboxypeptidases A and B in womb body tumors

The interrelationship between activity of trypsin-like proteases, carboxypeptidases A and B and the estrogen level in the womb body tissues has been studied. Results of correlation analysis suggest the existence of induction of trypsin-like proteases by estrogens; these proteases activate carboxypeptidases A and B from their zymogens. It has been shown, that carboxypeptidases may play essential role in the malignization process.     

Intra- versus extracellular effects of microglia-derived cysteine proteases in a conditioned medium transfer model

Activated microglia release inflammatory mediators that display either beneficial or harmful effects on neuronal survival and signaling. In the present study we demonstrate that exposure to lipopolysaccharide leads to an increase in the lysosomal cysteine proteases, cathepsin B, K, S, and X, in culture supernatants of the microglia cell line BV-2. In addition, we observed an up-regulation of cathepsins in the cytoplasmic fraction in response to stimulation with lipopolysaccharide. Conditioned medium from these cells was toxic to the neuroblastoma cell line Neuro2a. Experiments with membrane-permeable and membrane-impermeable cysteine protease inhibitors suggested that blocking extracellular cathepsins had no effect on microglia-mediated neuron death in this medium transfer model. However, intracellular cathepsins seem to trigger the release of neurotoxic factors. In lipopolysaccharide-stimulated BV-2 cells, inhibition of intracellular cathepsins significantly diminished microglial activation characterized by reduced expression of different proinflammatory cytokines, thereby reducing the neurotoxic effects of the medium. This hitherto unknown intracellular effect of cysteine proteases in activated microglia might connect chronic neuroinflammation with neurodegeneration.     

In silico Analysis of Sequential,Structural and Functional Diversity of Wheat Cystatins and Its Implication in Plant Defense

Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases.Cereal crops like wheat are continuously threatened by a multitude of pathogens,therefore cystatins offer to play a pivotal role in deciding the plant response.In order to study the need of having diverse specificities and activities of various cystatins,we conducted comparative analysis of six wheat cystatins(WCs) with twelve rice,seven barley,one sorghum and ten corn cystatin sequences employing different bioinformatics tools.The obtained results identified highly conserved signature sequences in all the cystatins considered.Several other motifs were also identified,based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering.Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins.Protein-protein interaction of WCs with papain supported the notion that functional diversity is a consequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs.Thus there is a significant conservation at the sequential and structural levels;however,concomitant variations maintain the functional diversity in this protein family,which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.     

豆梨半胱氨酸蛋白酶抑制剂基因的克隆及胁迫表达

采用RT-PCR结合RACE技术,从中国梨砧木——豆梨成熟种子中克隆获得豆梨半胱氨酸蛋白酶抑制剂基因(PcCPI)的cDNA全长序列,并通过半定量RT-PCR分析不同胁迫处理对该基因表达水平的影响.结果表明:(1)PcCPIcDNA序列长度为980 bp,开放阅读框78~812 bp,编码的多肽由信号肽(29个氨基酸)和成熟肽(216个氨基酸)组成,具有3个植物半胱氨酸蛋白酶抑制剂基因家族的高保守特征序列模式——LARFAVQEHN、QX-VXG和YQAKVWVKPW.(2)该蛋白与蔷薇科植物的CPI蛋白处于系统发育树的同一分支,且与苹果CPI蛋白的一致性最高(95.9%).(3)半定量RT-PCR结果显示:PcCPI基因在豆梨叶片中为诱导型表达,在人工模拟逆境条件下(包括喷施50μmol/L MeJA或100μmol/L ABA、刀片2次机械损伤2、00 mmol/L NaCl胁迫、4℃低温或30℃高温胁迫)处理4 h后,豆梨叶片中PcCPI基因表达量明显上升,表明该基因参与了豆梨对生物或非生物胁迫的防御机制.     

Purification and properties of proteases produced byalternaria alternata (Fr.) Keissl,regulation of production by fructose

A strain ofAlternaria alternata (Fr.) Keissl, when grown on wheat bran Czapek Dox medium was found to secrete one neutral and two alkaline proteases. The purified enzymes were found to be endo peptidases, the alkaline proteases being serine proteases and neutral proteases being cysteine proteases. Fructose when added to the culture medium was found to give rise to a new neutral protease at the expense of the neutral protease produced in the absence of fructose and was also found to enhance the production of alkaline proteases. It also appears that fructose modifies the alkaline proteases with respect to some characteristics such asV _max, Ea etc. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis indicated a significantly altered protein profile in fructose supplemented medium.     

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.     

Activation of Cysteine Proteases in Cowpea Plants during the Hypersensitive Response-A Form of Programmed Cell Death

There is increasing evidence that the hypersensitive response during plant–pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea–cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa.In vitroandin vivoprotease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response.     

Cysteine proteases: mode of action and role in epidermal differentiation

Desquamation or cell shedding in mammalian skin is known to involve serine proteases, aspartic proteases and glycosidases. In addition, evidence continues to accumulate that papain-like cysteine proteases and an inhibitor cystatin M/E largely confined to the cutaneous epithelia also play key roles in the process. This involves the complete proteolysis of cell adhesive structures of the stratum corneum, the corneodesmosomes and notably of the desmogleins. Continual cell replacement in the epidermis is the result of the balance between the loss of the outer squames and mitosis of the cells in the basal cell layer. This article provides a brief account of the salient features of the characteristics and catalytic mechanism of cysteine proteases, followed by a discussion of the relevant epidermal biology. The proteases include the asparaginyl endopeptidase legumain, which exerts a strict specificity for the hydrolysis of asparaginyl bonds, cathepsin-V and cathepsin-L. The control of these enzymes by cystatin M/E regulates the processing of transglutaminases and is crucial in the biochemical pathway responsible for regulating the cross-linking and desquamation of the stratum corneum. In addition, caspase-14 has now been shown to play a major part in epidermal maturation. Uncontrolled proteolytic activity leads to abnormal hair follicle formation and deleterious effects on the skin barrier function.     

Role of the Liver in Regulation of Body Cysteine and Taurine Levels: A Brief Review

The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome.     

Cysteine peptidases from Phytomonas serpens: biochemical and immunological approaches

Phytomonas serpens , a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi . In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens , while a strong 50-kDa cysteine peptidase was observed in T. cruzi . Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens . The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens , including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection.     

Cysteine Racemization in Peptide Synthesis: A New and Easy Detection Method

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris- alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine –peptides. Baseline enantiomeric resolution of theD ,L -S-β-(4-pyridylethyl)cysteine, and thus quantification ofD - enantiomer contents at levels ≤1%, is easily achieved by capillary zone electrophoresis exploiting the host–guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.     

Characterization and distribution of chymotrypsin-like and other digestive proteases in Colorado potato beetle larvae

Chymotrypsin-like, carboxypeptidase A-like and leucine aminopeptidase-like activities have been detected in the midgut of Colorado potato beetle larvae, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), in addition to the previously identified cathepsin B, D, and H. We have characterized a new chymotrypsin-like activity using the specific substrates N- succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide and N-benzoyl-L-tyrosine p-nitroanilide. This novel proteinase, with a pH optimum of 5.5–6.5, was neither activated by thiol compounds nor inhibited by cysteine proteinase inhibitors. Among several serine proteinase inhibitors tested, PMSF was the most effective. Gelatin-containing SDS-PAGE gels and activity staining after gel electrophoresis indicated that chymotrypsin-like activity was associated with a major band of about 63 Kda and a minor band of about 100 Kda. The major exopeptidases found in the larval midgut extracts were leucine aminopeptidase and carboxypeptidase A. Most endo- and exoproteolytic activities studied were evenly distributed among the midgut sections, indicating that there is no clear regional differentiation in the digestion of proteins. Chymotrypsin and cathepsin B, D, and H were mainly located in the endoperitrophic and ectoperitrophic spaces, with only a small activity associated with the midgut epithelium. In contrast, leucine aminopeptidase was mainly located on the wall tissue, although some activity was distributed between the ecto- and endoperitrophic spaces. The potential roles of Colorado potato beetle digestive chymotrypsin in the proteolytic activation of the δ-endotoxin from Bacillus thuringiensis, and in the use of protease inhibitors to disrupt protein digestion, are discussed. Arch. Insect Biochem. Physiol. 36:181–201, 1997. © 1997 Wiley-Liss, Inc.     

Effect of protease inhibitors and clenbuterol on theIn vitro degradation of dystrophin by endogenuous proteases in human skeletal muscle

Thein vitro degradation of dystrophin protein by endogenous proteases in human skeletal muscle has been investigated using a tissue homogenate assay system with subsequent protein analysis via SDS polyacrylamide electrophoresis and immunoblotting (using a monoclonal antibody to the central rod region of dystrophin). The rate of dystrophin degradation and nature of the proteolytic fragments formed at pH 5.5 and pH 7.5 (corresponding to the two major protease groups of relevance to intracellular protein catabolism) were broadly similar; incorporation of protease inhibitors in the above system suggested that Ca²⁺ activated proteinase and cathepsin D are principally responsible for the degradation of dystrophin at pH 7.5 and pH 5.5 respectively. The rate of dystrophin degradation at pH 7.5 was reduced by approximately 20% in the presence of 10^–5 M clenbuterol, a -adrenoceptor agonist with therapeutic potential in the treatment of human muscle wasting diseases.     

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.     

原位杂交检测斯氏肺吸虫童虫半胱氨酸蛋白酶的基因表达部位

目的 从基因水平研究半胱氨酸蛋白酶基因在斯氏肺吸虫童虫的表达,并进行虫体定位。方法 利用地高辛标记原位杂交技术检测半胱氨酸蛋白酶基因在斯氏肺吸虫童虫的表达并定位。结果 在斯氏肺吸虫童虫的肠管上皮细胞中呈强阳性着色,在虫体皮层细胞中有弱阳性着色。结论 半胱氨酸蛋白酶主要在斯氏肺吸虫童虫的肠管上皮细胞中表达,在虫体皮层细胞中也有弱表达。     

Cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity

Phytopathogenic bacteria use the type-III secretion system (TTSS) to inject effector proteins into plant cells, presumably to colonize their hosts. The function of these proteins inside plant cells has remained a mystery for years. The recent discovery that the effectors XopD, AvrXv4, AvrPphB, and AvrRpt2 have cysteine protease functions reveals that the proteolysis of host substrates is an important strategy employed by pathogens to alter plant physiology. Moreover, the characterization of these proteases and their targets provides new insight to mechanisms of bacterial virulence and the activation of plant immunity.