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Sample records for cathepsins

  1. Cathepsin D

    Czech Academy of Sciences Publication Activity Database

    Fusek, M.; Mareš, Michael; Větvička, V.

    Amsterdam : Academic Press, 2013 - (Salvesen, G.), s. 54-63 ISBN 978-0-12-382219-2 R&D Projects: GA AV ČR IAA400550705 Institutional support: RVO:61388963 Keywords : cathepsin D * protease * aspartic peptidase * apoptosis * cancer Subject RIV: CE - Biochemistry

  2. Cathepsins mediate tumor metastasis

    Institute of Scientific and Technical Information of China (English)

    Gong-Jun; Tan; Zheng-Ke; Peng; Jin-Ping; Lu; Fa-Qing; Tang

    2013-01-01

    Cathepsins are highly expressed in various human cancers, associated with tumor metastasis. It is superfamily, concluding A, B, C, D, E, F, G, H, L, K, O, S, V, and W family members. As a group of lysosomal proteinases or endopeptidases, each member has a different function, playing different roles in distinct tumorigenic processes such as proliferation, angiogenesis, metastasis, and invasion. Cathepsins belong to a diverse number of enzyme subtypes, including cysteine proteases, serine proteases and aspartic proteases. The contribution of cathepsins to invasion in human cancers is well documented, although the precise mechanisms by which cathepsins exert their effects are still not clear. In the present review, the role of cathepsin family members in cancer is discussed.

  3. Cathepsin proteases in Toxoplasma gondii

    OpenAIRE

    Dou, Zhicheng; Carruthers, Vern B.

    2011-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and n...

  4. Cathepsin Protease Inhibition Reduces Endometriosis Lesion Establishment.

    Science.gov (United States)

    Porter, Kristi M; Wieser, Friedrich A; Wilder, Catera L; Sidell, Neil; Platt, Manu O

    2016-05-01

    Endometriosis is a gynecologic disease characterized by the ectopic presence of endometrial tissue on organs within the peritoneal cavity, causing debilitating abdominal pain and infertility. Current treatments alleviate moderate pain symptoms associated with the disorder but exhibit limited ability to prevent new or recurring lesion establishment and growth. Retrograde menstruation has been implicated for introducing endometrial tissue into the peritoneal cavity, but molecular mechanisms underlying attachment and invasion are not fully understood. We hypothesize that cysteine cathepsins, a group of powerful extracellular matrix proteases, facilitate endometrial tissue invasion and endometriosis lesion establishment in the peritoneal wall and inhibiting this activity would decrease endometriosis lesion implantation. To test this, we used an immunocompetent endometriosis mouse model and found that endometriotic lesions exhibited a greater than 5-fold increase in active cathepsins compared to tissue from peritoneal wall or eutopic endometrium, with cathepsins L and K specifically implicated. Human endometriosis lesions also exhibited greater cathepsin activity than adjacent peritoneum tissue, supporting the mouse results. Finally, we tested the hypothesis that inhibiting cathepsin activity could block endometriosis lesion attachment and implantation in vivo. Intraperitoneal injection of the broad cysteine cathepsin inhibitor, E-64, significantly reduced the number of attached endometriosis lesions in our murine model compared to vehicle-treated controls demonstrating that cathepsin proteases contribute to endometriosis lesion establishment, and their inhibition may provide a novel, nonhormonal therapy for endometriosis. PMID:26482207

  5. [Cathepsin K antagonists: preclinical and clinical data].

    Science.gov (United States)

    Gamsjäger, Marion; Resch, Heinrich

    2015-02-01

    Cathepsin K, a cysteine protease, is an essential enzyme in degradation of collagen type I. Since cathepsin K is relatively specific to osteoclasts, it represents a promising candidate for drug development. In the past decades, efforts have been made in developing highly potent, selective and orally applicable cathepsin K inhibitors. In contrast to balicatib and relacatib, whose drug development programmes were stopped due to cutaneous side-effects related to limited drug specificity, the more specific cathepsin K inhibitors odanacatib (ODN) and ONO-5334 have entered clinical trials. Odanacatib progressively increases bone mineral density (BMD) and decreases bone resorption markers in postmenopausal women with low BMD. Its clinical efficacy and safety was confirmed by several clinical studies but indicates that odanacatib is characterized by a resolution-of-effect with increases in bone resorption and rapid decreases in BMD following treatment discontinuation. A phase III fracture prevention study in postmenopausal women with osteoporosis is currently in the final phase. PMID:25572547

  6. Cathepsin K: a unique collagenolytic cysteine peptidase.

    Science.gov (United States)

    Novinec, Marko; Lenarčič, Brigita

    2013-09-01

    Cathepsin K has emerged as a promising target for the treatment of osteoporosis in recent years. Initially identified as a papain-like cysteine peptidase expressed in high levels in osteoclasts, the important role of this enzyme in bone metabolism was highlighted by the finding that mutations in the CTSK gene cause the rare recessive disorder pycnodysostosis, which is characterized by severe bone anomalies. At the molecular level, the physiological role of cathepsin K is reflected by its unique cleavage pattern of type I collagen molecules, which is fundamentally different from that of other endogenous collagenases. Several cathepsin K inhibitors have been developed to reduce the excessive bone matrix degradation associated with osteoporosis, with the frontrunner odanacatib about to successfully conclude Phase 3 clinical trials. Apart from osteoclasts, cathepsin K is expressed in different cell types throughout the body and is involved in processes of adipogenesis, thyroxine liberation and peptide hormone regulation. Elevated activity of cathepsin K has been associated with arthritis, atherosclerosis, obesity, schizophrenia, and tumor metastasis. Accordingly, its activity is tightly regulated via multiple mechanisms, including competitive inhibition by endogenous macromolecular inhibitors and allosteric regulation by glycosaminoglycans. This review provides a state-of-the-art description of the activity of cathepsin K at the molecular level, its biological functions and the mechanisms involved in its regulation. PMID:23629523

  7. Relationship between Cathepsin B, Cathepsin D Expression and Biological Behaviour in Mucinous Colorectal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    王娅兰; 林晓

    2004-01-01

    @@ The proteolytic enzymes secreted by cancer cells are believed to play an important role in cancer invasion and metastasis. In this study, cathepsin B (CB)and cathepsin D (CD) were detected by method of immunohistochemistry in tissue specimens 48 samples of human colorectal carcinomas.

  8. Cathepsin B gene disruption induced Leishmania donovani proteome remodeling implies cathepsin B role in secretome regulation.

    Directory of Open Access Journals (Sweden)

    Teklu Kuru Gerbaba

    Full Text Available Leishmania cysteine proteases are potential vaccine candidates and drug targets. To study the role of cathepsin B cysteine protease, we have generated and characterized cathepsin B null mutant L. donovani parasites. L. donovani cathepsin B null mutants grow normally in culture, but they show significantly attenuated virulence inside macrophages. Quantitative proteome profiling of wild type and null mutant parasites indicates cathepsin B disruption induced remodeling of L. donovani proteome. We identified 83 modulated proteins, of which 65 are decreased and 18 are increased in the null mutant parasites, and 66% (55/83 of the modulated proteins are L. donovani secreted proteins. Proteins involved in oxidation-reduction (trypanothione reductase, peroxidoxins, tryparedoxin, cytochromes and translation (ribosomal proteins are among those decreased in the null mutant parasites, and most of these proteins belong to the same complex network of proteins. Our results imply virulence role of cathepsin B via regulation of Leishmania secreted proteins.

  9. Cathepsin E Promotes Pulmonary Emphysema via Mitochondrial Fission

    OpenAIRE

    Zhang, Xuchen; Shan, Peiying; Homer, Robert; Zhang, Yi; Petrache, Irina; Mannam, Praveen; Lee, Patty J.

    2014-01-01

    Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose funct...

  10. Immunohistochemical study on expression of cathepsin in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Wan Long Lin; Cong Jia Chen; Han Gao Zhou

    2000-01-01

    AIM To study the expression of cathepsin B in gastric carcinoma and its relationship with pathologic type.METHODS The cathepsin B expression in 54 specimens of human gastric adenocarcinoma was studied byimmunohistochemistry.RESULTS The cathepsin B expression was detected in 33/54 (61.1%) specimens of human gastriccarcinoma and in 3/54 (5.6%) of normal tissue (P<0.01). There was no obvious correlation between theexpression of cathepsin B and pathologic type of gastric adenocarcinoma.CONCLUSION There is a high expression of cathepsin B in human gastric adenocarcinoma.

  11. xtraction and Characterization of Cathepsin Inhibitor from Milkfish

    Directory of Open Access Journals (Sweden)

    Tati Nurhayati

    2015-06-01

    Full Text Available Abstract Proteolytic enzyme is distributed acros all organism including fish. Cysteine proteases are the largest group of proteolytic enzyme. Lysosomal cathepsin, one of cysteine protease enzyme, cause softening and degradation of myofibril protein and it’s activity is regulated by endogenous inhibitors. The purposes of this study were to optimize the extraction cathepsin inhibitors from the skin, muscles, and viscera of fish, to partially purify the cathepsin inhibitors of selected sources, and to study the characteristics of the cathepsin inhibitor. The cathepsin inhibitor could be extracted from muscle fish and partially purified using ammonium sulfate of 70%. The purified cathepsin inhibitor had optimum temperature at 40°C and the optimum at pH 8. Metal ions decreased the activity of the protease inhibitor, except 1 mM of metal ion Mn2+ and Na+.

  12. Cathepsins and cystatin C in atherosclerosis and obesity.

    Science.gov (United States)

    Lafarge, Jean-Charles; Naour, Nadia; Clément, Karine; Guerre-Millo, Michèle

    2010-11-01

    Given the increasing prevalence of human obesity worldwide, there is an urgent need for a better understanding of the molecular mechanisms linking obesity to metabolic and cardiovascular diseases. Our knowledge is nevertheless limited regarding molecules linking adipose tissue to downstream complications. The importance of cathepsins was brought to light in this context. Through a large scale transcriptomic analysis, our group recently identified the gene encoding cathepsin S as one of the most deregulated gene in the adipose tissue of obese subjects and positively correlated with body mass index. Other members of the cathepsin family are expressed in the adipose tissue, including cathepsin K and cathepsin L. Given their implication in atherogenesis, these proteases could participate into the well established deleterious relationship between enlarged adipose tissue and increased cardiovascular risk. Here, we review the clinical and experimental evidence relevant to the role of cathepsins K, L and S and their most abundant endogenous inhibitor, cystatin C, in atherosclerosis and in obesity. PMID:20417681

  13. Differential impact of cysteine cathepsins on genetic mouse models of de novo carcinogenesis: cathepsin B as emerging therapeutic target

    Directory of Open Access Journals (Sweden)

    ThomasReinheckel

    2012-07-01

    Full Text Available Lysosomal cysteine cathepsins belong to a family of 11 human proteolytic enzymes. Some of them correlate with progression in a variety of cancers and therefore are considered as potential therapeutic targets. Until recently, the contribution of individual cathepsins to tumorigenesis and tumor progression remained unknown. By crossing various types of mouse cancer models with mice where specific cathepsins have been ablated, we contributed to this gap of knowledge and will summarize the results in this report. The employed models are the Rip1-Tag2 model for pancreatic neuroendocrine tumors, the K14-HPV16 model for squamous skin and cervical cancers, and the MMTV-PyMT model for metastasizing breast cancer, the KPC model for pancreatic ductal adenocarcinoma, and the APCmin mice developing early stages of intestinal neoplasia. All models harbor mutations in relevant tumor suppressors and/or cell-type specific expression of potent oncogenes, which initiate de novo carcinogenesis in the targeted tissues. In all these models deletion of cathepsin B led to suppression of the aggressiveness of the respective cancer phenotype. Cathepsin B may network with other proteases as it was shown for cathepsin X/Z. In contrast, deletion of cathepsin L was beneficial in the RiP1-Tag2 model, but enhanced tumorigenesis in the APCmin, and the K14-HPV16 mice. A logical consequence of these results would be to further pursue selective inhibition of cathepsin B. Moreover, it became clear that cathepsins B and S derived from cells of the tumor microenvironment support cancer growth. Strikingly, delivery of broad spectrum cysteine cathepsin inhibitors in the tumor microenvironment disrupts the permissive ecosystem of the cancer and results in impaired growth or even in regression of the tumor. In addition, combination of cysteine cathepsin inhibition and standard chemotherapy improves the therapeutic response of the latter.

  14. Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages.

    Science.gov (United States)

    Pires, David; Marques, Joana; Pombo, João Palma; Carmo, Nuno; Bettencourt, Paulo; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2016-01-01

    Cathepsins are proteolytic enzymes that function in the endocytic pathway, especially in lysosomes, where they contribute directly to pathogen killing or indirectly, by their involvement in the antigen presentation pathways. Mycobacterium tuberculosis (MTB) is a facultative intracellular pathogen that survives inside the macrophage phagosomes by inhibiting their maturation to phagolysosomes and thus avoiding a low pH and protease-rich environment. We previously showed that mycobacterial inhibition of the proinflammatory transcription factor NF-κB results in impaired delivery of lysosomal enzymes to phagosomes and reduced pathogen killing. Here, we elucidate how MTB also controls cathepsins and their inhibitors, cystatins, at the level of gene expression and proteolytic activity. MTB induced a general down-regulation of cathepsin expression in infected cells, and inhibited IFNγ-mediated increase of cathepsin mRNA. We further show that a decrease in cathepsins B, S and L favours bacterial survival within human primary macrophages. A siRNA knockdown screen of a large set of cathepsins revealed that almost half of these enzymes have a role in pathogen killing, while only cathepsin F coincided with MTB resilience. Overall, we show that cathepsins are important for the control of MTB infection, and as a response, it manipulates their expression and activity to favour its intracellular survival. PMID:27572605

  15. Development of nitrile-based peptidic inhibitors of cysteine cathepsins.

    Science.gov (United States)

    Frizler, Maxim; Stirnberg, Marit; Sisay, Mihiret Tekeste; Gütschow, Michael

    2010-01-01

    It is now becoming clear that several papain-like cysteine cathepsins are involved in the pathophysiology of diseases such as osteoporosis, autoimmune disorders, and cancer. Therefore, the development of potent and selective cathepsin inhibitors is an attractive subject for medicinal chemists. New advances have been made for nitrile-based inhibitors, leading to the identification of the cathepsin K inhibitor odanacatib and other candidates with potential for therapeutic use. This review summarizes the development of peptidic and peptidomimetic compounds with an electrophilic nitrile 'warhead' as inhibitors of the cysteine cathepsins B, S, L, C, and K. Peptide nitriles have been shown to reversibly react with the active site cysteine under formation of a covalent thioimidate adduct. The structural optimization with respect to the positions P3, P2, P1, P1', and P2' resulted in the identification of potent and selective inhibitors of the corresponding cathepsins. The underlying structure-activity relationships are discussed herein. PMID:20166952

  16. Plasma levels of cathepsins L, K, and V and risks of abdominal aortic aneurysms

    DEFF Research Database (Denmark)

    Lv, Bing-Jie; Lindholt, Jes S; Wang, Jing;

    2013-01-01

    Cathepsin L (CatL), cathepsin K (CatK), and cathepsin V (CatV) are potent elastases implicated in human arterial wall remodeling. Whether plasma levels of these cathepsins are altered in patients with abdominal aortic aneurysms (AAAs) remains unknown....

  17. Cathepsins are required for Toll-like receptor 9 responses

    International Nuclear Information System (INIS)

    Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9

  18. Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity.

    Science.gov (United States)

    Falgueyret, Jean-Pierre; Desmarais, Sylvie; Oballa, Renata; Black, W Cameron; Cromlish, Wanda; Khougaz, Karine; Lamontagne, Sonia; Massé, Frederic; Riendeau, Denis; Toulmond, Sylvie; Percival, M David

    2005-12-01

    The lysosomal cysteine protease cathepsin K is a target for osteoporosis therapy. The aryl-piperazine-containing cathepsin K inhibitor CRA-013783/L-006235 (1) displays greater than 4000-fold selectivity against the lysosomal/endosomal antitargets cathepsin B, L, and S. However, 1 and other aryl-piperazine-containing analogues, including balicatib (10), are approximately 10-100-fold more potent in cell-based enzyme occupancy assays than against each purified enzyme. This phenomenon arises from their basic, lipophilic nature, which results in lysosomal trapping. Consistent with its lysosomotropic nature, 1 accumulates in cells and in rat tissues of high lysosome content. In contrast, nonbasic aryl-morpholino-containing analogues do not exhibit lysosomotropic properties. Increased off-target activities of basic cathepsin K inhibitors were observed in a cell-based cathepsin S antigen presentation assay. No potency increases of basic inhibitors in a functional cathepsin K bone resorption whole cell assay were detected. Therefore, basic cathepsin K inhibitors, such as 1, suffer from reduced functional selectivities compared to those predicted using purified enzyme assays. PMID:16302795

  19. Caught in the act: the crystal structure of cleaved cathepsin L bound to the active site of Cathepsin L.

    Science.gov (United States)

    Sosnowski, Piotr; Turk, Dušan

    2016-04-01

    Cathepsin L is a ubiquitously expressed papain-like cysteine protease involved in the endosomal degradation of proteins and has numerous roles in physiological and pathological processes, such as arthritis, osteoporosis, and cancer. Insight into the specificity of cathepsin L is important for elucidating its physiological roles and drug discovery. To study interactions with synthetic ligands, we prepared a presumably inactive mutant and crystallized it. Unexpectedly, the crystal structure determined at 1.4 Å revealed that the cathepsin L molecule is cleaved, with the cleaved region trapped in the active site cleft of the neighboring molecule. Hence, the catalytic mutant demonstrated low levels of catalytic activity. PMID:26992470

  20. Expression of Cathepsins B, D, and G in Infantile Hemangioma

    OpenAIRE

    Itinteang, Tinte; Chudakova, Daria A.; Jonathan C. Dunne; Davis, Paul F.; Tan, Swee T.

    2015-01-01

    Aims The role of the renin–angiotensin system (RAS) in the biology of infantile hemangioma (IH) represents an emerging paradigm, particularly the involvement of renin, angiotensin converting enzyme, and angiotensin II. This study investigated the expression of cathepsins B, D, and G, enzymes that may modulate the RAS, in IH. Materials and Methods The expression of cathepsins B, D, and G was examined using immunohistochemistry, enzyme activity assays, mass spectrometry, and NanoStri...

  1. Effect of heavy metal cations on the activity of cathepsin D (in vitro study) Effect of heavy metal cations on the activity of cathepsin D (in vitro study)

    OpenAIRE

    Alicja Karwowska; Radosław Łapiński; Marek Gacko; Ewa Grzegorczyk; Joanna Żurawska; Jan K. Karczewski

    2012-01-01

    We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity of
    cathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobin
    was the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cations
    inhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these organelles.We studied the effect of heavy metal c...

  2. Silver and Gold Nanoparticles Alter Cathepsin Activity In vitro

    Science.gov (United States)

    Speshock, Janice L.; Braydich-Stolle, Laura K.; Szymanski, Eric R.; Hussain, Saber M.

    2011-12-01

    Nanomaterials are being incorporated into many biological applications for use as therapeutics, sensors, or labels. Silver nanomaterials are being utilized for biological implants and wound dressings as an antiviral material, whereas gold nanomaterials are being used as biological labels or sensors due to their surface properties and biocompatibility. Cytotoxicity data of these materials are becoming more prevalent; however, little research has been performed to understand how the introduction of these materials into cells affects cellular processes. Here, we demonstrate the impact that silver and gold nanoparticles have on cathepsin activity in vitro. Cathepsins are important cellular proteases that are imperative for proper immune system function. We have selected to examine gold and silver nanoparticles due to the increased use of these materials in biological applications. This manuscript depicts how both of these types of nanomaterials affect cathepsin activity, which could impact the host's immune system and its ability to respond to pathogens. Cathepsin B activity decreases in a dose-dependent manner with all nanoparticles tested. Alternatively, the impact of nanoparticles on cathepsin L activity depends greatly on the type and size of the material.

  3. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporothioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection.Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide(ASODN)targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000.The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls.The expression of cathepsin B mRNA was examined by RT-PCR an...

  4. Effect of heavy metal cations on the activity of cathepsin D (in vitro study Effect of heavy metal cations on the activity of cathepsin D (in vitro study

    Directory of Open Access Journals (Sweden)

    Alicja Karwowska

    2012-10-01

    Full Text Available We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity of
    cathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobin
    was the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cations
    inhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these organelles.We studied the effect of heavy metal cations: Fe 2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ on the activity of
    cathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobin
    was the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg2+ cations
    inhibit the activity of cathepsin D. Cations Hg2+ damage lysosomes and release cathepsin D from these organelles.

  5. Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose.

    Directory of Open Access Journals (Sweden)

    Minjeong Jung

    Full Text Available Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.

  6. Human cathepsin L, a papain-like collagenase without proline specificity.

    Science.gov (United States)

    Korenč, Matevž; Lenarčič, Brigita; Novinec, Marko

    2015-11-01

    Several members of the papain-like peptidase family have the ability to degrade collagen molecules by cleaving within the triple helix region of this difficult substrate. A common denominator of these peptidases is their ability to cleave substrates with Pro in the P2 position. In humans, cathepsin K is the best-known papain-like collagenase. Here, we investigate the collagenolytic activity of human cathepsin L, which is closely related to cathepsin K. We show that, despite lacking proline specificity, cathepsin L efficiently cleaves type I collagen within the triple helix region and produces a cleavage pattern similar to that of cathepsin K. We demonstrate that both enzymes have similar affinities for type I collagen and are able to release proteolytic fragments from insoluble collagen. Moreover, cathepsin K is only approximately fourfold more potent than cathepsin L in releasing fragments from reconstituted fibrils of FITC-labeled collagen. Replacing active site residues of cathepsin L with those from cathepsin K introduces cathepsin K-like specificity towards synthetic substrates and increases the collagenolytic activity of cathepsin L. Replacing three residues in the S2 subsite is sufficient to produce a mutant with collagenolytic activity on par with human cathepsin K. These results provide a basis for engineering collagenolytic activity into non-collagenolytic papain-like scaffolds. PMID:26306868

  7. Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore.

    Science.gov (United States)

    Frizler, Maxim; Yampolsky, Ilia V; Baranov, Mikhail S; Stirnberg, Marit; Gütschow, Michael

    2013-09-21

    An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE. PMID:23912233

  8. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  9. IL-8 and cathepsin B as melanoma serum biomarkers.

    Science.gov (United States)

    Zhang, Hongtao; Fu, Ting; McGettigan, Suzanne; Kumar, Suresh; Liu, Shujing; Speicher, David; Schuchter, Lynn; Xu, Xiaowei

    2011-01-01

    Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality. Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically. However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests. Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05). More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality. Our study provides a novel approach to examine serum biomarkers. PMID:21673904

  10. Cathepsin-D And Tnf-α in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    T. Salman

    1996-01-01

    Full Text Available In a study of 34 normal healthy controls, 35 patients with urinary tract bilharziasis and 93 bladder cancer patients (62 of them are operable cases and 31 are non-operable ones, serum tumor necrosis factor alpha (TNF-α and cytosolic Cathepsin-D were estimated. Though both potential markers were elevated in bladder cancer patients, neither Cathepsin-D nor TNF-α showed associations of prognostic value since there were no positive correlations with tumor stages, grades or association of tumors with bilharzia ova or lymph node involvement.

  11. Effect of heavy metal cations on the activity of cathepsin D (in vitro study).

    Science.gov (United States)

    Karwowska, Alicja; Łapiński, Radosław; Gacko, Marek; Grzegorczyk, Ewa; Żurawska, Joanna; Karczewski, Jan K

    2012-01-01

    We studied the effect of heavy metal cations: Fe²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺ on the activity of cathepsin D in human aorta homogenate and blood serum. The concentration of cations was 1 mmol/l. Hemoglobin was the cathepsin D substrate. The activity of cathepsin D was determined at pH 3.5. Only Hg²⁺ cations inhibit the activity of cathepsin D. Cations Hg²⁺ damage lysosomes and release cathepsin D from these organelles. PMID:23042275

  12. The Effect of Washing and Inhibitor on Cathepsin Activity of Silver Carp

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The effect of washing and temperature on the activity of cathepsins of Silver carp was studied.The result showed that the activity of cathepsin L was higher than those of cathepsin B and H.The total catalysis activity of these three enzymes was the highest at 55℃ after washing.The inhibiting effect of soybean protein and potato starch on cathepsin L also had been studied,the results showed that soybean protein and potato starch could decrease activity of cathepsins L significantly.

  13. Cathepsin L inactivates human trypsinogen whereas cathepsin L deletion reduces the severity of pancreatitis in mice

    Science.gov (United States)

    Wartmann, Thomas; Mayerle, Julia; Kähne, Thilo; Sahin-Tóth, Miklós; Ruthenbürger, Manuel; Matthias, Rainer; Kruse, Anne; Reinheckel, Thomas; Peters, Christoph; Weiss, F. Ulrich; Sendler, Matthias; Hans-Lippert; Schulz, Hans-Ulrich; Aghdassi, Ali; Dummer, Annegret; Teller, Steffen; Halangk, Walter; Lerch, Markus M.

    2010-01-01

    Background & Aims Acute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL), the second most abundant lysosomal cysteine proteinase, can also process trypsinogen to active trypsin and has a role in pancreatitis. Methods In CTSL-deficient (Ctsl−/−) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl−/− and Ctsb−/− mice were studied. Results CTSL was expressed in human and mouse pancreas, where it colocalized with trypsinogen in secretory vesicles and lysosomes and was secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl−/− mice, compared with wild-type controls, whereas apoptosis and intrapancreatic trypsin activity were increased in Ctsl−/− mice. CTSL induced cleavage of trypsinogen occurred 3 amino acids toward the C terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (TAP). This elongated TAP was not detected by ELISA but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic. Conclusions CTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity. PMID:19900452

  14. Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

    OpenAIRE

    Andrade, Sheila Siqueira; Gouvea, Iuri Estrada; Silva, Mariana Cristina C.; Castro, Eloísa Dognani; de Paula, Cláudia A. A.; Okamoto, Debora; Oliveira, Lilian; Peres, Giovani Bravin; Ottaiano, Tatiana; Facina, Gil; Nazário, Afonso Celso Pinto; Campos, Antonio Hugo J. F. M.; Paredes-Gamero, Edgar Julian; Juliano, Maria; da Silva, Ismael D. C. G.

    2016-01-01

    Background Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes t...

  15. Construction and transfection of sense/antisense eukaryotic expression vectors for human cathepsin L gene

    Institute of Scientific and Technical Information of China (English)

    Maolin He; Anmin Chen

    2005-01-01

    Objective: To obtain sense/antisense eukaryotic expression vectors for human cathepsin L gene, and study the biological effects on human osteosarcoma cell line MG-63 after transfection. Methods: Cathepsin L gene sense/antisense eukaryotic expression vectors were constructed with recombinant technology and transfected into the human osteosarcoma cell line MG-63. The expression of cathepsin L gene mRNA was examined with RT-PCR and the expression of cathepsin L was examined with Western blot. Results: The sense/antisense recombinant eukaryotic expression vectors for cathepsin L were successfully constructed and transfected into MG-63 cell.Conclusion: Antisense cathepsin L gene can significantly inhibit the expression of cathepsin L mRNA and protein.

  16. Complex modulation of peptidolytic activity of cathepsin D by sphingolipids

    Czech Academy of Sciences Publication Activity Database

    Žebrakovská, Iva; Máša, Martin; Srp, Jaroslav; Horn, Martin; Vávrová, K.; Mareš, Michael

    2011-01-01

    Roč. 1811, č. 12 (2011), s. 1097-1104. ISSN 1388-1981 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506 Keywords : sphingolipid * phospholipid * inhibition * activation * cathepsin D * enzyme regulation Subject RIV: CE - Biochemistry Impact factor: 5.269, year: 2011

  17. Cathepsin X in serum from patients with colorectal cancer

    DEFF Research Database (Denmark)

    Vizin, Tjasa; Christensen, Ib Jarle; Nielsen, Hans Jørgen;

    2012-01-01

    Up-regulation of lysosomal cysteine protease cathepsin X (Cat X) is associated with disorders of the immune system and neurodegenerative diseases, while its role in the development and progression of cancer is less understood. Enhanced secretion of pro-Cat X was observed in malignant processes, a...

  18. A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica.

    Science.gov (United States)

    Cancela, Martín; Acosta, Daniel; Rinaldi, Gabriel; Silva, Edileusa; Durán, Rosario; Roche, Leda; Zaha, Arnaldo; Carmona, Carlos; Tort, Jose F

    2008-10-01

    Secreted cysteine proteases are relevant actors in parasite biology, taking part in critical host colonization roles such as traversing tissue barriers, immune evasion and nutrient digestion. In the trematode Fasciola hepatica, the initial step to successful infection of the mammalian host is the excystment of metacercariae and the invasion through the intestinal wall by the newly excysted juveniles (NEJ). While the cathepsin L-like cysteine proteinases secreted by the adult fluke have been extensively characterized, the cataloguing and description of the cathepsins B and L reported in the invasive stages is only sketchy. To identify the cathepsins expressed during excystment and early invasion we constructed cDNA libraries encoding NEJ cathepsins B and L. We found two cathepsin L-like cysteine proteinases (CL3, CL4) and three cathepsins B (CB1, CB2, CB3) which are predominantly expressed in NEJ. Phylogenetic analysis showed that NEJ-expressed cathepsins L constitute a well-defined clade separate from the adult enzymes. Excystment induction resulted in a significant increment in activity towards cathepsin-specific fluorogenic substrates in metacercariae homogenates, consistent with the detection of precursor and mature forms of cathepsins B and L before and after induction. In NEJ culture supernatants, protein and relative activity profiles show subtle changes during the first 48 h, with prevalence of cathepsin L-like activity, although cathepsins CB3 and CL3 were detected by mass spectrometry. Noticeably, the hydrolysis of a substrate with proline in the P2 position was predominant, a property only shared with adult CL2 and vertebrate cathepsin K among the C1A subfamily of cysteine proteases. Collectively these mRNA, protein and enzymatic data demonstrate the existence of a NEJ-specific repertoire of cathepsins expressed early in invasion, distinct to those used by other trematodes, potentially relevant for specific vaccine and chemotherapy design. The diversity

  19. Cathepsin L knockdown enhances curcumin-mediated inhibition of growth, migration, and invasion of glioma cells.

    Science.gov (United States)

    Fei, Yao; Xiong, Yajie; Zhao, Yifan; Wang, Wenjuan; Han, Meilin; Wang, Long; Tan, Caihong; Liang, Zhongqin

    2016-09-01

    Curcumin can be used to prevent and treat cancer. However, its exact underlying molecular mechanisms remain poorly understood. Cathepsin L, a lysosomal cysteine protease, is overexpressed in several cancer types. This study aimed to determine the role of cathepsin L in curcumin-mediated inhibition of growth, migration, and invasion of glioma cells. Results revealed that the activity of cathepsin L was enhanced in curcumin-treated glioma cells. Cathepsin L knockdown induced by RNA interference significantly promoted curcumin-induced cytotoxicity, apoptosis, and cell cycle arrest. The knockdown also inhibited the migration and invasion of glioma cells. Our results suggested that the inhibition of cathepsin L can enhance the sensitivity of glioma cells to curcumin. Therefore, cathepsin L may be a new target to enhance the efficacy of curcumin against cancers. PMID:27373979

  20. Pharmacological and genetic evidence that cathepsin B is not the physiological activator of rodent prorenin.

    Science.gov (United States)

    Percival, M David; Toulmond, Sylvie; Coulombe, Nathalie; Cromlish, Wanda; Desmarais, Sylvie; Liu, Susana; St-Jacques, René; Gauthier, Jacques Yves; Fournier, Jean-Francois

    2010-12-01

    Renin is the first enzyme in the renin-angiotensin-aldosterone system which is the principal regulator of blood pressure and hydroelectrolyte balance. Previous studies suggest that cathepsin B is the activator of the prorenin zymogen. Here, we show no difference in plasma renin activity, or mean arterial blood pressure between wild-type and cathepsin B knockout mice. To account for potential gene compensation, a potent, selective, reversible cathepsin B inhibitor was developed to determine the role of cathepsin B on prorenin processing in rats. Pharmacological inhibition of cathepsin B in spontaneously hypertensive and double transgenic rats did not result in a reduction in renal mature renin protein levels or plasma renin activity. We conclude that cathepsin B does not play a significant role in this process in rodents. PMID:20868234

  1. Synthesis and Biochemical Evaluation of Thiochromanone Thiosemicarbazone Analogues as Inhibitors of Cathepsin L

    Science.gov (United States)

    2012-01-01

    A series of 36 thiosemicarbazone analogues containing the thiochromanone molecular scaffold functionalized primarily at the C-6 position were prepared by chemical synthesis and evaluated as inhibitors of cathepsins L and B. The most promising inhibitors from this group are selective for cathepsin L and demonstrate IC50 values in the low nanomolar range. In nearly all cases, the thiochromanone sulfide analogues show superior inhibition of cathepsin L as compared to their corresponding thiochromanone sulfone derivatives. Without exception, the compounds evaluated were inactive (IC50 > 10000 nM) against cathepsin B. The most potent inhibitor (IC50 = 46 nM) of cathepsin L proved to be the 6,7-difluoro analogue 4. This small library of compounds significantly expands the structure–activity relationship known for small molecule, nonpeptidic inhibitors of cathepsin L. PMID:24900494

  2. IL8 and Cathepsin B as Melanoma Serum Biomarkers

    Directory of Open Access Journals (Sweden)

    Xiaowei Xu

    2011-02-01

    Full Text Available Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality. Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically. However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests. Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05. More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality. Our study provides a novel approach to examine serum biomarkers.

  3. Mining a cathepsin inhibitor library for new antiparasitic drug leads.

    Science.gov (United States)

    Ang, Kenny K H; Ratnam, Joseline; Gut, Jiri; Legac, Jennifer; Hansell, Elizabeth; Mackey, Zachary B; Skrzypczynska, Katarzyna M; Debnath, Anjan; Engel, Juan C; Rosenthal, Philip J; McKerrow, James H; Arkin, Michelle R; Renslo, Adam R

    2011-01-01

    The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ∼ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts. PMID:21572521

  4. Calpain and cathepsin activities in post mortem fish and meat muscles

    OpenAIRE

    Cheret, Romuald; Delbarre Ladrat, Christine; De Lamballerie Anton, Marie; VERREZ-BAGNIS Veronique

    2007-01-01

    Post mortem tenderization is one of the most unfavourable quality changes in fish muscle and this contrasts with muscle of mammalian meats. The tenderization can be partly attributed to the acid lysosomal cathepsins and cytosolic neutral calcium-activated calpains. In this study, these proteases from fish and bovine muscles were quantified and compared. The cathepsin B and L activities were in more important amounts in sea bass white muscle than in bovine muscle. On the other hand, cathepsin ...

  5. Usefulness of Serum Cathepsin L as an Independent Biomarker in Patients With Coronary Heart Disease

    OpenAIRE

    Liu, Yingxian; Li, Xiangping; Peng, Daoquan; Tan, Zheng; Liu, Hongmin; Qing, Yingnan; Xue, Yanqiong; Shi, Guo-Ping

    2008-01-01

    Higher levels of cysteinyl cathepsin L were detected in human atherosclerotic lesions than in healthy aortas. However, a link between human coronary heart disease (CHD) and systemic cathepsin L levels remains unknown. A total of 137 volunteers with diagnosed acute and previous myocardial infarction (MI) and stable and unstable angina pectoris in addition to 48 controls were asked to undergo coronary angiography. Serum cathepsin L, high-sensitivity C-reactive protein, fasting glucose, and lipi...

  6. Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

    OpenAIRE

    Takio, K; Towatari, T; Katunuma, N.; Teller, D C; Titani, K

    1983-01-01

    The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage ...

  7. P61CATHEPSIN K IN AN IN VITRO MODEL OF GLIOMA ANGIOGENESIS

    OpenAIRE

    Briggs, S.; K. Stevenson; Verbovšek, U.; Yin, L.H.; Pilkington, G.; Lah, T.; Fillmore, H.L.

    2014-01-01

    INTRODUCTION: Cathepsin K, a cysteine protease expressed in osteoclasts, involved in bone resorption is expressed in other cells including brain cells. Reports suggest that cathepsin K may be involved in cancers associated with bone metastasis. Little is known about its expression in brain tumours. There is evidence of a potential interaction of cathepsin K with stromal cell derived factor 1 (SDF-1) in haemapoietic stem cell motility. Because of the importance of SDF-1 in brain tumour angioge...

  8. Cathepsin activity and texture in Atlantic salmon muscle

    OpenAIRE

    Sheng, Yuancheng

    2015-01-01

    Cathepsins, a family of lysosomal proteases, are believed to play a role in muscle tenderization. In the present study the activity of cathapsin B+L in Atlantic salmon muscle and a possible influence on the textural quality was studied. Total of 98 Atlantic salmon from 10 families were slaughtered and pre-rigor filleted. This salmon fillet texture was measured instrumentally at 5 days post-mortem. The cathapsin activities were measured on muscle samples frozen immediately after slaughter. Sta...

  9. The Clinical Significance of Cathepsin S Expression in Human Astrocytomas

    OpenAIRE

    Flannery, Thomas; Gibson, David; Mirakhur, Menakshi; McQuaid, Stephen; Greenan, Caroline; Trimble, Anne; Walker, Brian; McCormick, Derek; Johnston, Patrick G.

    2003-01-01

    Early local invasion by astrocytoma cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that Cat...

  10. Cathepsin B inactivation attenuates hepatic injury and fibrosis during cholestasis

    OpenAIRE

    Canbay, Ali; Guicciardi, Maria Eugenia; Higuchi, Hajime; Feldstein, Ariel; Bronk, Steven F.; Rydzewski, Robert; Tanai, Makiko; Gores, Gregory J.

    2004-01-01

    Although a lysosomal, cathepsin B–dependent (Ctsb-dependent) pathway of apoptosis has been described, the contribution of this pathway to tissue damage remains unclear. Our aim was to ascertain if Ctsb inactivation attenuates liver injury, inflammation, and fibrogenesis after bile duct ligation (BDL). In 3-day BDL mice, hepatocyte apoptosis, mitochondrial cytochrome c release, and serum alanine aminotransferase (ALT) values were reduced in Ctsb–/– versus Ctsb+/+ animals. Likewise, R-3032 (a C...

  11. Structure of a Kunitz-type potato cathepsin D inhibitor

    Czech Academy of Sciences Publication Activity Database

    Guo, J.; Erskine, P. T.; Coker, A. R.; Wood, S. P.; Cooper, J. B.; Mareš, Michael; Baudyš, Miroslav

    2015-01-01

    Roč. 192, č. 3 (2015), s. 554-560. ISSN 1047-8477 R&D Projects: GA ČR GA15-18929S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : potato cathepsin D inhibitor * Kunitz-type protease inhibitor * protein X-ray structure * reactive-site loop * docking Subject RIV: CE - Biochemistry Impact factor: 3.231, year: 2014

  12. Cathepsin b: a potential prognostic marker for inflammatory breast cancer

    OpenAIRE

    Cavallo-Medved Dora; Shaalan Mohamed A; El-Shinawi Mohamed; Mohamed Mona M; Nouh Mohamed A; Khaled Hussein M; Sloane Bonnie F

    2011-01-01

    Abstract Background Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC. Methods In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IB...

  13. Endothelial cells and cathepsins: Biochemical and biomechanical regulation.

    Science.gov (United States)

    Platt, Manu O; Shockey, W Andrew

    2016-03-01

    Cathepsins are mechanosensitive proteases that are regulated not only by biochemical factors, but are also responsive to biomechanical forces in the cardiovascular system that regulate their expression and activity to participate in cardiovascular tissue remodeling. Their elastinolytic and collagenolytic activity have been implicated in atherosclerosis, abdominal aortic aneurysms, and in heart valve disease, all of which are lined by endothelial cells that are the mechanosensitive monolayer of cells that sense and respond to fluid shear stress as the blood flows across the surfaces of the arteries and valve leaflets. Inflammatory cytokine signaling is integrated with biomechanical signaling pathways by the endothelial cells to transcribe, translate, and activate either the cysteine cathepsins to remodel the tissue or to express their inhibitors to maintain healthy cardiovascular tissue structure. Other cardiovascular diseases should now be included in the study of the cysteine cathepsin activation because of the additional biochemical cues they provide that merges with the already existing hemodynamics driving cardiovascular disease. Sickle cell disease causes a chronic inflammation including elevated TNFα and increased numbers of circulating monocytes that alter the biochemical stimulation while the more viscous red blood cells due to the sickling of hemoglobin alters the hemodynamics and is associated with accelerated elastin remodeling causing pediatric strokes. HIV-mediated cardiovascular disease also occurs earlier in than the broader population and the influence of HIV-proteins and antiretrovirals on endothelial cells must be considered to understand these accelerated mechanisms in order to identify new therapeutic targets for prevention. PMID:26458976

  14. Acridone alkaloids as potent inhibitors of cathepsin V.

    Science.gov (United States)

    Severino, Richele P; Guido, Rafael V C; Marques, Emerson F; Brömme, Dieter; da Silva, M Fátima das G F; Fernandes, João B; Andricopulo, Adriano D; Vieira, Paulo C

    2011-02-15

    Cathepsin V is a lysosomal cysteine peptidase highly expressed in thymus, testis and corneal epithelium. Eleven acridone alkaloids were isolated from Swinglea glutinosa (Bl.) Merr. (Rutaceae), with eight of them being identified as potent and reversible inhibitors of cathepsin V (IC(50) values ranging from 1.2 to 3.9 μM). Detailed mechanistic characterization of the effects of these compounds on the cathepsin V-catalyzed reaction showed clear competitive inhibition with respect to substrate, with dissociation constants (K(i)) in the low micromolar range (2, K(i)=1.2 μM; 6, K(i)=1.0 μM; 7, K(i)=0.2 μM; and 11, K(i)=1.7 μM). Molecular modeling studies provided important insight into the structural basis for binding affinity and enzyme inhibition. Experimental and computational approaches, including biological evaluation, mode of action assessment and modeling studies were successfully employed in the discovery of a small series of acridone alkaloid derivatives as competitive inhibitors of catV. The most potent inhibitor (7) has a K(i) value of 200 nM. PMID:21277783

  15. Changes of cathepsin B in human photoaging skin both in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    LAI Wei; ZHENG Yue; YE Zhang-zhang; SU Xiang-yang; WAN Miao-jian; GONG Zi-jian; XIE Xiao-yuan; LIU Wei

    2010-01-01

    Background Cathepsin B plays an important role in cell cycle, extracellular matrix changes and cutaneous tumorigenesis: whether it plays a role in photoaged skin remains unknown. This study aimed to investigate the role of cathepsin B in skin photoaging in vivo and in vitro. Methods The expressions of cathepsin B were compared with immunohistochemical methods in solar exposed skin and solar protected skin of six healthy Chinese volunteers. The mRNA and protein expression of cathepsin B in ultraviolet light A (UVA) induced premature senescence fibroblasts in vitro were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting technique. Results Decreased expression of cathepsin B was observed in photoaged skin compared with that of the solar protected skin. In the UVA induced, premature senescence fibroblasts, a lower expression of cathepsin B was detected by Western blotting and a decreased synthesis of cathepsin B mRNA in the same cells was revealed by real-time RT-PCR. Conclusions The results demonstrated a significant negative correlation between skin photoaging and cathepsin B in vitro and in vivo. We propose that cathepsin B, besides matrix metalloproteinases and antioxidant enzymes, is involved in the process of skin photoaging in that it contributes to extracellular matrix remodelling and is a dominant protease in cellular apoptosis and senescence.

  16. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    International Nuclear Information System (INIS)

    Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression

  17. P61CATHEPSIN K IN AN IN VITRO MODEL OF GLIOMA ANGIOGENESIS

    Science.gov (United States)

    Briggs, S.; Stevenson, K.; Verbovšek, U.; Yin, L.H.; Pilkington, G.; Lah, T.; Fillmore, H.L.

    2014-01-01

    INTRODUCTION: Cathepsin K, a cysteine protease expressed in osteoclasts, involved in bone resorption is expressed in other cells including brain cells. Reports suggest that cathepsin K may be involved in cancers associated with bone metastasis. Little is known about its expression in brain tumours. There is evidence of a potential interaction of cathepsin K with stromal cell derived factor 1 (SDF-1) in haemapoietic stem cell motility. Because of the importance of SDF-1 in brain tumour angiogenesis and recruitment of glioma like stem cells to vascular niches, we investigated cathepsin K in an in vitro model of angiogenesis. METHOD: Brain endothelial cells (hCMEC) and glioma cell lines (SNB-19 and UP-007) cultured under normoxic and hypoxic conditions were analysed using flow cytometry and western blotting. Angiogenesis was assessed using an in vitro model of brain endothelial cell tube formation. Brain endothelial tube length, number of tube projections and number of branch points were measured. RESULTS: Under hypoxic conditions, there is a significant decrease in cathepsin K expression in brain endothelial cells when compared to normoxic conditions (P ≤ 0.05). Addition of Odanacatib, a cathepsin K inhibitor, to the angiogenesis assay revealed that inhibition of cathepsin K resulted in a significant increase in endothelial tube length in normoxic conditions (p < 0.05). CONCLUSION: The decrease in cathepsin K expression in endothelial cells under hypoxia, coupled with the increase in tube length following inhibition of cathepsin K, suggests an involvement of cathepsin K with angiogenesis. These data provide rationale and basis for further study into the function of cathepsin K and its relationship with SDF-1 in gliomas.

  18. Serum cathepsin H as a potential prognostic marker in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Schweiger, A; Christensen, Ib Jarle; Nielsen, Hans Jørgen; Sørensen, Steen; Brunner, N; Kos, J

    2005-01-01

    Cathepsin H is a lysosomal cysteine protease that may participate in tumor progression. In order to evaluate its potential as a prognostic marker, its protein levels were measured by ELISA in preoperative sera from 324 patients with colorectal cancer. The level of cathepsin H was significantly...

  19. Bone Microenvironment Modulates Expression and Activity of Cathepsin B in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Izabela Podgorski

    2005-03-01

    Full Text Available Prostate cancers metastasize to bone leading to osteolysis. Here we assessed proteolysis of DOcollagen I (a bone matrix protein and, for comparison, DO-collagen IV, by living human prostate carcinoma cells in vitro. Both collagens were degraded, this degradation was reduced by inhibitors of matrix metallo, serine, cysteine proteases. Because secretion of the cysteine protease cathepsin B is increased in human breast fibroblasts grown on collagen I gels, we analyzed cathepsin B levels and secretion in prostate cells grown on collagen I gels. Levels and secretion were increased only in DU145 cells-cells that expressed the highest baseline levels of cathepsin B. Secretion of cathepsin B was also elevated in DU145 cells grown in vitro on human bone fragments. We further investigated the effect of the bone microenvironment on cathepsin B expression and activity in vivo in a SCID-human model of prostate bone metastasis. High levels of cathepsin B protein and activity were found in DU145, PC3, LNCaP bone tumors, although the PC3 and LNCaP cells had exhibited low cathepsin B expression in vitro. Our results suggest that tumor-stromal interactions in the context of the bone microenvironment can modulate the expression of the cysteine protease cathepsin B.

  20. Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.

    Science.gov (United States)

    Cox, Jennifer M; Troutt, Jason S; Knierman, Michael D; Siegel, Robert W; Qian, Yue-Wei; Ackermann, Bradley L; Konrad, Robert J

    2012-11-15

    There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology. PMID:22922382

  1. Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer

    NARCIS (Netherlands)

    Edgington-Mitchell, L.E.; Rautela, J.; Duivenvoorden, H.M.; Jayatilleke, K.M.; Linden, W.A. van der; Verdoes, M.; Bogyo, M.; Parker, B.S.

    2015-01-01

    Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvi

  2. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

    DEFF Research Database (Denmark)

    Parker, Erica N; Song, Jiangli; Kishore Kumar, G D;

    2015-01-01

    selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5μ...

  3. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA

    Science.gov (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

  4. The effect of cathepsin K deficiency on airway development and TGF-β1 degradation

    Directory of Open Access Journals (Sweden)

    Saftig Paul

    2011-05-01

    Full Text Available Background Cathepsin K, a cysteine protease predominantly expressed in osteoclasts, is a major drug target for the treatment of osteoporosis. Recent findings, however, indicate that cathepsin K is also involved in non-skeletal metabolism. The development of fibrotic phenotypes in lung and skin is a concern for cathepsin K inhibitors presently evaluated in clinical trials. Cathepsin K is expressed in lung tissue and has been implicated in lung fibrosis. However, little is known about the role of cathepsin K in airway development and its effect on TGF-β1 degradation. Methods We investigated the effects of cathepsin K-deficiency on alterations in airway integrity, extracellular matrix composition, and TGF-β1 expression and degradation. Lung homogenates of wild-type and cathepsin K-deficient mice were used to evaluate their contents of collagen, glycosaminoglycans, and TGF-β1. The accessibility of TGF-β1 to cathepsin K-mediated degradation was determined in vitro and lung fibroblast proliferations in wild-type and cathepsin K-deficient cells were evaluated. Results Lung airway cathepsin K expression in wild-type mice remained constant between 1 and 6 months of age and the airway integrity was maintained. In contrast, after 2 months of age, all Ctsk-/- mice demonstrated increased airway epithelium thickness by 16-28%, a lower structural airway integrity (1-2 score units lower, elevated cytokeratin expression of 12%, increased α-actin and vimentin expression by 50% and 70%, increased area of smooth muscle cells by 15%, elevated hydroxyproline and GAGs content by 20% and 25%, and increased TGF-β1 expression by 25%. TGF-β1 proved an efficient substrate of cathepsin K and TGF-β1 protein content in lung was increased by a potent cathepsin inhibitor. Lung fibroblasts from Ctsk-/- mice after TGF-β1 treatment showed increased proliferation rates, increased levels of TGF-β1 by 30%, and increased ECM secretion. Conclusion This study suggests that

  5. Increased Circulating Cathepsin K in Patients with Chronic Heart Failure

    OpenAIRE

    Guangxian Zhao; Yuzi Li; Lan Cui; Xiang Li; Zhenyi Jin; Xiongyi Han; Ennan Fang; Yihua Gao; Dongmei Zhou; Haiying Jiang; Xueying Jin; Guanghao Piao; Xiangshan Li; Guang Yang; Jiyong Jin

    2015-01-01

    Cysteinyl cathepsin K (CatK) is one of the most potent mammalian collagenases involved in cardiovascular disease. Here, we investigated the clinical predictive value of serum CatK levels in patients with chronic heart failure (CHF). We examined 134 patients with CHF, measuring their serum CatK, troponin I, high-sensitive C-reactive protein, and pre-operative N-terminal pro-brain natriuretic peptide levels. The patients were divided into two groups: the 44 patients who showed a left ventricula...

  6. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L.

    Science.gov (United States)

    Parker, Erica N; Song, Jiangli; Kishore Kumar, G D; Odutola, Samuel O; Chavarria, Gustavo E; Charlton-Sevcik, Amanda K; Strecker, Tracy E; Barnes, Ashleigh L; Sudhan, Dhivya R; Wittenborn, Thomas R; Siemann, Dietmar W; Horsman, Michael R; Chaplin, David J; Trawick, Mary Lynn; Pinney, Kevin G

    2015-11-01

    Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre

  7. Lack of cathepsin activities alter or prevent the development of lung granulomas in a mouse model of sarcoidosis

    Directory of Open Access Journals (Sweden)

    Percival M David

    2011-01-01

    Full Text Available Abstract Background Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis. Methods Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor. Results When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed. Conclusions Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.

  8. CORRELATION BETWEEN LAMININ AND CATHEPSIN D EXPRESSIONS IN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    CHEN Feng; CHEN Wei-hong; ZHENG Jian-ming; HUANG Ling

    2006-01-01

    Objective: Laminin is a major glycoprotein component of basement membrance which is an important barrier to tumor cells which must be breeched before metastatic spread can occur. Proteolytic enzymes play an important role in mediating the passage of cancer cells through the basement membrane (BM) and extracellular matrix. We compared the patterns of laminin and cathepsin D (CD) expressions in a range of benign and malignant breast lesions to better understand the process of tumor progression. Methods: One hundred and sixty-two cases of breast samples comprising 18 fibroadeomas, 22 cases of fibrocystic disease, 96 cases of invasive ductal carcinoma and 26 carcinomas with intraductal components were evaluated for laminin and cathepsin D expressions by immunohistochemical staining. Results: The prevalence of CD positivity in both neoplastic and stromal cell components were significantly higher in higher histological grade tumors compared to lower grades (P<0.001). Various severity of BM disruption correlated with histological grade of the carcinomas (P<0.001). There was a negative correlation between the laminin expression and CD presence. Conclusion: In the process of cancer cell invasion and metastasis, the basement membrane is disrupted by proteinase secreted by cancer cells, especially by stroma cells of cancer.

  9. Immunohistochemical expression of cathepsin L in atopic dermatitis and lichen planus

    Directory of Open Access Journals (Sweden)

    Zeinab A El Ashmawy

    2015-01-01

    Full Text Available Background: Cathepsin L is a member of papain superfamily. It seems to promote T-cell survival, selection maturation in the thymus and enhance the antigen presentation. Cathepsin L plays an important role in tumor necrosis factors (TNF-α induced cell death. Also it degrades the tight junction between cornedesomses in the epidermis. Elevated expression of cathepsin L has been found in many inflammatory and neoplastic diseases. Objective: The aim of this study was to determine immunohistochemical expression of cathepsin L in atopic dermatitis (AD and lichen planus (LP patients in order to evaluate its role in the pathogenesis of both diseases. Materials and Methods: This study included 15 patients with AD (Group I, 15 patients with LP (Group II, in addition to 10 healthy skin specimens served as controls (Group III. Punch biopsies were taken from lesional skin of the patients and controls for immunohistochemical detection of cathepsin L expression. Results: Highly significant increase was found in cathepsin L expression in AD and LP patients compared to controls [P = 0.001]. Conclusion: Cathepsin L could be implicated as an important protease in the pathogenesis of AD and LP. It could be a useful marker for assessing AD severity.

  10. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    He Maolin; Xiao Zengming; Li Shide; Chen Anmin

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporotbioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection. Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide (ASODN) targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000. The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls. The expression of cathepsin B mRNA was examined by RT-PCR and the expression of cathepsin B was examined by Western blot. The invasive capability of MG-63 cells was evaluated by the boydern chamber assay. Results The expression of cathcpsin B was obviously inhibited in antlsense oligodeoxynucleotide treated cells compared with the control cells. The number of invading MG-63 cells was significantly lower in the ASODN-treated groups than that in the control groups. Conclusion The cathepsin B ASODN significantly inhibits the expression of cathepsin B and invasive ability of MG-63 cell in osteosarcoma.

  11. Cocaine potentiates cathepsin B secretion and neuronal apoptosis from HIV-infected macrophages.

    Science.gov (United States)

    Zenón, Frances; Segarra, Annabell C; Gonzalez, Mariangeline; Meléndez, Loyda M

    2014-12-01

    Substance abuse is a risk factor for HIV infection and progression to AIDS. Recent evidence establishes that cocaine use promotes brain perivascular macrophage infiltration and microglia activation. The lysosomal protease cathepsin B is increased in monocytes from patients with HIV dementia and its secretion induces 10-15% of neurotoxicity. Here we asked if cocaine potentiates cathepsin B secretion from HIV-infected monocyte-derived macrophages (MDM) and its effect in neuronal apoptosis. Samples of plasma, CSF, and post-mortem brain tissue from HIV positive patients that used cocaine were tested for cathepsin B and its inhibitors to determine the in vivo relevance of these findings. MDM were inoculated with HIV-1ADA, exposed to cocaine, and the levels of secreted and bioactive cathepsin B and its inhibitors were measured at different time-points. Cathepsin B expression (p cocaine treated MDM compared with HIV-infected cocaine negative controls. Increased levels of cystatin B expression was also found in supernatants from HIV-cocaine treated MDM (p cocaine users over non-drug users. Our results demonstrated that cocaine potentiates cathepsin B secretion in HIV-infected MDM and increase neuronal apoptosis. These findings provide new evidence that cocaine synergize with HIV-1 infection in increasing cathepsin B secretion and neurotoxicity. PMID:25209871

  12. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    Science.gov (United States)

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression. PMID:23667900

  13. A furanquinone from Paulownia tomentosa stem for a new cathepsin K inhibitor.

    Science.gov (United States)

    Park, Youmie; Kong, Jae Yang; Cho, Heeyeong

    2009-10-01

    In the search for novel inhibitors of cathepsin K, a new furanquinone compound, methyl 5-hydroxy-dinaphtho[1,2-2'3']furan-7,12-dione-6-carboxylate (1a), showed in vitro inhibitory activities for cathepsin K. Compound 1a was isolated originally from Paulownia tomentosa stem and its derivatives were synthesized. Furanquinone compounds (1a, 1b, 1c and 1d) were also found to be capable of inhibiting cathepsin L, which is closely related to cathepsin K. The inhibitory activity of the parent compound 1a (IC50 = 21 microm) for cathepsin K was slightly higher than those of the other three derivatives that have a methoxy (1b), propoxy (1c) or acetoxy (1d) group (IC50 = 33-66 microm) in the 5-position of compound 1a. This implies that the 5-hydroxyl functional group of 1a may have favorable effects on the reduction potential which are related to the cathepsin K inhibitory activities of furanquinone compounds. Therefore, the cathepsin K inhibitory activity of a new furanquinone compound is proposed. PMID:19277969

  14. Mutant K-ras Regulates Cathepsin B Localization on the Surface of Human Colorectal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Dora Cavallo-Medved

    2003-11-01

    Full Text Available Cathepsin B protein and activity are known to localize to the basal plasma membrane of colon carcinoma cells following the appearance of K-ras mutations. Using immunofluorescence and subcellular fractionation techniques and two human colon carcinoma cell lines—one with a mutated K-ras allele (HCT 116 and a daughter line in which the mutated allele has been disrupted (HKh-2—we demonstrate that the localization of cathepsin B to caveolae on the surface of these carcinoma cells is regulated by mutant K-ras. In HCT 116 cells, a greater percentage of cathepsin B was distributed to the caveolae, and the secretion of cathepsin B and pericellular (membrane-associated and secreted cathepsin B activity were greater than observed in HKh-2 cells. Previous studies established the light chain of annexin II tetramer, p11, as a binding site for cathepsin B on the surface of tumor cells. The deletion of active K-ras in HKh-2 cells reduced the steady-state levels of p11 and caveolin-1 and the distribution of pl1 to caveolae. Based upon these results, we speculate that cathepsin B, a protease implicated in tumor progression, plays a functional role in initiating proteolytic cascades in caveolae as downstream components of this cascade (e.g., urokinase plasminogen activator and urokinase plasminogen activator receptor are also present in HCT 116 caveolae.

  15. Usefulness of Serum Cathepsin L as an Independent Biomarker in Patients With Coronary Heart Disease

    Science.gov (United States)

    Liu, Yingxian; Li, Xiangping; Peng, Daoquan; Tan, Zheng; Liu, Hongmin; Qing, Yingnan; Xue, Yanqiong; Shi, Guo-Ping

    2009-01-01

    Higher levels of cysteinyl cathepsin L were detected in human atherosclerotic lesions than in healthy aortas. However, a link between human coronary heart disease (CHD) and systemic cathepsin L levels remains unknown. A total of 137 volunteers with diagnosed acute and previous myocardial infarction (MI) and stable and unstable angina pectoris in addition to 48 controls were asked to undergo coronary angiography. Serum cathepsin L, high-sensitivity C-reactive protein, fasting glucose, and lipid protein profiles were measured. Serum cathepsin L levels were significantly higher in patients with CHD than in those without CHD (p <0.001). The significance persisted after adjusting for most major confounders. Patients with unstable angina pectoris had higher serum cathepsin L levels than those with stable angina pectoris (p = 0.02). Of patients with acute coronary syndrome, those with acute MI had higher serum cathepsin L levels than those with unstable angina pectoris (p <0.05) and patients with previous MI had the highest levels. Importantly, serum cathepsin L associated positively with number of coronary branch luminal narrowings (R = 0.38, p <0.001), Gensini scores (R = 0.44, p <0.001), high-sensitivity C-reactive protein (R = 0.32, p <0.001), fasting glucose (R = 0.16, p <0.03), and cigarette smokers (R = 0.27, p <0.001), but inversely with high-density lipoprotein (R = -0.23, p = 0.002) and apolipoprotein A1 (R = -0.19, p = 0.01) in all subjects. In conclusion, after adjusting for these confounders, we found that serum cathepsin L correlated positively and independently with Gensini score, suggesting that serum cathepsin L serves as a novel and independent biomarker for CHD. PMID:19195505

  16. Role of cathepsin E in decidual macrophage of patients with recurrent miscarriage.

    Science.gov (United States)

    Goto, Shinobu; Ozaki, Yasuhiko; Suzumori, Nobuhiro; Yasukochi, Atsushi; Kawakubo, Tomoyo; Furuno, Tadahide; Nakanishi, Mamoru; Yamamoto, Kenji; Sugiura-Ogasawara, Mayumi

    2014-05-01

    In a previous study, we reported that the cathepsin-cystatin system caused endometrial dysfunction in early pregnancy. Here, we investigated the existence and contribution of cathepsin E in early pregnancy in patients with recurrent miscarriage (RM). The effect of cathepsin deficiency on fertility and female reproductive organs were also analyzed in CatE(-/-) mice. Human studies were conducted in a hospital setting, with informed consent. Cervical mucus was collected from RM patients in early pregnancy (4-6 gestational weeks, n = 21), and the pregnancy outcome was compared prospectively. The cathepsin E expression in decidua of RM patients (n = 49) and normal pregnant women undergoing elective surgical abortion (n = 24) was measured using SDS-PAGE, and western blot analysis. Decidual macrophages were isolated from RM patients (n = 6) and stimulated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Results from the mouse model showed that CatE(-/-) mice were fertile, but the litter number was significantly smaller. The uterus of CatE(-/-) mice showed granulation tissue. In human samples, protease activity of cathepsin E measured with Fluorescence-Quenching Substrate (KYS-1) in cervical mucus of patients who developed miscarriage was markedly decreased compared with patients without RM. The expression of cathepsin E in decidua, semi-quantified by SDS-PAGE, western blot analysis was significantly lower in RM patients compared with patients without RM. By double staining immunofluorescence, the staining of cathepsin E was observed in CD14 or CD68 positive cells in all deciduas. Upon stimulation with LPS and IFN-γ, the expression of cathepsin E in cell lysate of decidual macrophages was markedly reduced in RM patients compared with controls. The results suggested that decreased activity of cathepsin E produced by decidual macrophages might be responsible for the induction of miscarriages in some RM patients. PMID:24464956

  17. Downregulation of uPAR and Cathepsin B Retards Cofilin Dephosphorylation

    OpenAIRE

    Christopher S Gondi; Kandhukuri, Neelima; Kondraganti, Shakuntala; Gujrati, Meena; Olivero, William C.; Dinh, Dzung H.; Rao, Jasti S.

    2006-01-01

    Cathepsin B and uPAR play key roles in cancer cell migration and invasion. Here, we demonstrate that the simultaneous, siRNA-mediated downregulation of uPAR and cathepsin B inhibits glioma cell migration and is accompanied by cytoskeletal condensation. We show that the dephosphorylation of cofilin is inhibited by the downregulation of uPAR alone and, to a lesser extent, by the downregulation of cathepsin B alone, and that the effect was much higher by the downregulation of both molecules by p...

  18. Purification and characterization of cathepsin B from the skeletal muscles of agama stellio stellio

    International Nuclear Information System (INIS)

    1. Cathepsin b from the muscles of Jordanian lizard Agama stellio stellio was purified to homogeneity by a series of column chromatography on DEAE-sephadex, thio propyl sepharose and sephadex G-100 2. The molecular weight of cathepsin B isolated was to be 31800 dalton by using SDS-PAGE, and 33000 dalton by gel filtration, and its isoelectric point was measured to be 4.2 by isoelectric focusing. 3. Cathepsin B had ph optimum of 5.5, required a thiol-reducing reagent for activation and was inhibited by thiol-protease inhibitors. 4. The Km and K cat values for Z-Phe-Arg-Mca were determined to be 0.161mM and 238 S-1. 5. Cathepsin B acted on oligopeptide substrates mainly as di peptidyl carboxypeptidase. (authors). 22 refs., 3 tabs., 2 figs

  19. Plasma cathepsin S and cystatin C levels and risk of abdominal aortic aneurysm

    DEFF Research Database (Denmark)

    Lv, Bing-Jie; Lindholt, Jes Sanddal; Cheng, Xiang;

    2012-01-01

    Human abdominal aortic aneurysm (AAA) lesions contain high levels of cathepsin S (CatS), but are deficient in its inhibitor, cystatin C. Whether plasma CatS and cystatin C levels are also altered in AAA patients remains unknown....

  20. Cysteine protease cathepsins and matrix metalloproteinases in the development of abdominal aortic aneurysms

    OpenAIRE

    Qin, Yanwen; Cao, Xu; Yang, Yaoguo; Shi, Guo-Ping

    2013-01-01

    Both cysteine protease cathepsins and matrix metalloproteinases are implicated in the pathogenesis of abdominal aortic aneurysms (AAAs) in humans and animals. Blood and aortic tissues from humans or animals with AAAs contain much higher levels of these proteases, and often lower levels of their endogenous inhibitors, than do blood and aortic tissues from healthy subjects. Protease- and protease inhibitor-deficient mice and synthetic protease inhibitors have affirmed that cysteinyl cathepsins ...

  1. Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer

    OpenAIRE

    Edgington-Mitchell, Laura E.; Rautela, Jai; Duivenvoorden, Hendrika M.; Jayatilleke, Krishnath M.; Wouter A. van der Linden; Verdoes, Martijn; Bogyo, Matthew; Parker, Belinda S.

    2015-01-01

    Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvironment that function to promote tumor growth and invasion, such that they may be valid targets for anti-metastatic therapeutic approaches. Using activity-based probes, we have examined the activit...

  2. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    OpenAIRE

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and...

  3. Gene Targeting of the Cysteine Peptidase Cathepsin H Impairs Lung Surfactant in Mice

    OpenAIRE

    Bühling, Frank; Kouadio, Martin; Caroline E Chwieralski; Kern, Ursula; Hohlfeld, Jens M.; Klemm, Nicole; Friedrichs, Nicole; Roth, Wera; Deussing, Jan M.; Peters, Christoph; Reinheckel, Thomas

    2011-01-01

    Background The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh) has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B). Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant prot...

  4. Cocaine potentiates cathepsin B secretion and neuronal apoptosis from HIV-infected macrophages

    OpenAIRE

    Zenón, Frances; Segarra, Annabell C.; Gonzalez, Mariangeline; Meléndez, Loyda M.

    2014-01-01

    Substance abuse is a risk factor for HIV infection and progression to AIDS. Recent evidence establishes that cocaine use promotes brain perivascular macrophage infiltration and microglia activation. The lysosomal protease cathepsin B is increased in monocytes from patients with HIV dementia and its secretion induces 10-15% of neurotoxicity. Here we asked if cocaine potentiates cathepsin B secretion from HIV-infected monocyte-derived macrophages (MDM) and its effect in neuronal apoptosis. Samp...

  5. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: david.kirsch@duke.edu [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  6. Amyloid β-protein, Cystatin C and Cathepsin B as Biomarkers of Alzheimer's Disease

    OpenAIRE

    Sundelöf, Johan

    2010-01-01

    It is suggested that Alzheimer’s disease (AD) is caused by an imbalance between production, degradation and clearance of the amyloid-β (Aβ) protein. This imbalance leads to aggregation of Aβ and tau proteins and neurodegeneration in the brain. Today there is increasing evidence that the balance between the protease cathepsin B and the protease inhibitor cystatin C affects the tendency for Aβ to aggregate. The primary aim of this thesis was to investigate Aβ, cystatin C and cathepsin B levels ...

  7. Study of a Novel Antiosteoporosis Screening Model Targeted on Cathepsin K

    Institute of Scientific and Technical Information of China (English)

    JUN YANG; GUANG-DONG SHANG; YUE-QIN ZHANG

    2004-01-01

    To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E.coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. Results With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. Conclusions The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.

  8. Cathepsin B, L, and D activities in colorectal carcinomas: relationship with clinico-pathological parameters.

    Science.gov (United States)

    Adenis, A; Huet, G; Zerimech, F; Hecquet, B; Balduyck, M; Peyrat, J P

    1995-09-25

    Cathepsins, which are secreted by tumour and/or stromal cells, are thought to be involved in the degradative processes of tumour invasion and metastasis. The purpose of our study was to compare the cytosolic content of cathepsin B, L, and D in a series of matched malignant and adjacent normal colorectal tissues. Further we attempted to correlate these different proteinase values to classical clinico-pathological prognostic variables. Cathepsin B, L, and D activities were higher in tumour tissues than in normal mucosa (P B, L, and D activities either as a function of gender (except for cathepsin B values), age at time of surgery, tumour site, tumour differentiation, tumour stage (TNM or Astler-Coller staging system) or whether or not we found a mucinous component. Based on our data, cathepsin B seems to be the most discriminant parameter of the three proteinases that we studied, suggesting that cathepsin B expression may be of critical value in the progression of colorectal cancers. PMID:7585467

  9. Profilin 1 as a target for cathepsin X activity in tumor cells.

    Directory of Open Access Journals (Sweden)

    Urša Pečar Fonović

    Full Text Available Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

  10. Olomoucine inhibits cathepsin L nuclear translocation, activates autophagy and attenuates toxicity of 6-hydroxydopamine.

    Science.gov (United States)

    Fei, Xi-Feng; Qin, Zheng-Hong; Xiang, Bei; Li, Ling-Yun; Han, Feng; Fukunaga, Kohji; Liang, Zhong-Qin

    2009-04-01

    The finding of nuclear translocation of cathepsin L and its ability to process the CDP/Cux transcription factor uncovers an important role of cathepsin L in control of cell cycle progression. As the expression of certain cell cycle regulators is associated with nigral neuronal death, the present study was sought to investigate if nuclear translocation of cathepsin L and expression of certain cyclins were induced in DA neurons by 6-hydroxydopamine (6-OHDA). The neuroprotective effects of the cell cycle inhibitor olomoucine against 6-OHDA-induced death of nigral neurons were examined. Using immunocytochemistry and real-time PCR we demonstrated that cyclin D1, cyclin B1 and proliferating cell nuclear antigen (PCNA) were aberrantly expressed in some dopaminergic neurons after 6-OHDA infusion. The nuclear translocation of cathepsin L and up-regulation of LC3, a protein involved in autophagy, were observed in nigral DA neurons. Olomoucine, a cyclin dependent kinase (CDK) inhibitor, reduced contralateral rotations and the loss of TH-positive neurons in substantia nigra induced by lesion with 6-OHDA. Pretreatment of rats or primary DA neurons with olomoucine resulted in a partial blockade of nuclear translocation of cathepsin L. Olomoucine also increased the expression of punctate LC3 immunoreactivity, indicating activation of autophagy. These findings suggest that olomoucine may exert neuroprotective effects through inhibiting cathepsin L nuclear translocation and activating autophagy. PMID:19368812

  11. Relation of Cystatin C and Cathepsin B Expression to the Pathological Grade and Invasion of Human Gliomas

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    OBJECTIVE To explore the relation of cystatin C and cathepsin B expression to the pathological grade and invasion of human gliomas.METHODS A immunohistochemical method was used to detect the expression of cystatin C and cathepsin B in 57 glioma samples.RESULTS The expression of cystatin C in high-grade (Grade Ⅲ~Ⅳ )gliomas was significantly weaker than that in low-grade(Grade Ⅰ~Ⅱ, P=0.0001).On the other hand, the expression of cathepsin B in high-grade gliomas was significantly stronger than that in low-grade (P=0.0001). Cystatin C expression correlated inversely with cathepsin B expression in gliomas (P=0.01).CONCLUSION Cystatin C and cathepsin B expression is related to the pathological grade and invasion of gliomas. Combining detection of cystatin C and cathepsin B expressions might provide significant information for clinical assessment of maglignant phenotypes and invasion of gliomas.

  12. Cathepsin l expression is up-regulated by hypoxia in human melanoma cells: role of its 5`-untranslated region

    OpenAIRE

    Jean, Didier; Rousselet, Nathalie; Frade, Raymond

    2008-01-01

    Abstract Overexpression of cathepsin L, a cysteine protease and consequently procathepsin L secretion switch the phenotype of human melanoma cells to highly tumorigenic and strongly metastatic. This led us to identify the DNA regulatory sequences involved in the regulation of cathepsin L expression in highly metastatic human melanoma cells. Data demonstrated presence of inhibitory sequences in the 3? region downstream of cathepsin L gene and in the 3?- and 5?-flanking region of GC/...

  13. Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer.

    LENUS (Irish Health Repository)

    Sullivan, Shane

    2012-02-01

    Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

  14. Plasma cathepsin D isoforms and their active metabolites increase after myocardial infarction and contribute to plasma renin activity.

    Science.gov (United States)

    Naseem, R Haris; Hedegard, Wade; Henry, Timothy D; Lessard, Jennifer; Sutter, Kathryn; Katz, Stephen A

    2005-03-01

    Plasma renin activity (PRA) is often found to increase after myocardial infarction (MI). Elevated PRA may contribute to increased myocardial angiotensin II that is responsible for maladaptive remodeling of the myocardium after MI. We hypothesized that MI would also result in cardiac release of cathepsin D, a ubiquitous lysosomal enzyme with high renin sequence homology. Cathepsin D release from damaged myocardial tissue could contribute to angiotensin formation by acting as an enzymatic alternate to renin. We assessed circulating renin and cathepsin D from both control and MI patient plasma (7-20 hours after MI) using shallow gradient focusing that allowed for independent measurement of both enzymes. Cathepsin D was increased significantly in the plasma after MI (P < 0.001). Furthermore, circulating active cathepsin D metabolites were also significantly elevated after MI (P < 0.04), and contained the majority of cathepsin D activity in plasma. Spiking control plasma with cathepsin D resulted in a variable but significant (P = 0.005) increase in PRA using a clinical assay. We conclude that 7-20 hours after MI, plasma cathepsin D is significantly elevated and most of the active enzymatic activity is circulating as plasma metabolites. Circulating cathepsin D can falsely increase clinical PRA determinations, and may also provide an alternative angiotensin formation pathway after MI. PMID:15739123

  15. Silicatein α : Cathepsin L-Like Protein in Sponge Biosilica

    Science.gov (United States)

    Shimizu, Katsuhiko; Cha, Jennifer; Stucky, Galen D.; Morse, Daniel E.

    1998-05-01

    Earth's biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge Tethya aurantia, making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein α , β , and γ . The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein α , the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein α , although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein α also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.

  16. Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

    Science.gov (United States)

    Rieman, D J; McClung, H A; Dodds, R A; Hwang, S M; Holmes, M W; James, I E; Drake, F H; Gowen, M

    2001-03-01

    Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae. PMID:11248658

  17. Cathepsin b: a potential prognostic marker for inflammatory breast cancer

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    Cavallo-Medved Dora

    2011-01-01

    Full Text Available Abstract Background Inflammatory breast cancer (IBC is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC. Methods In this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1, a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups. Results Our results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC. Conclusions CTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.

  18. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    International Nuclear Information System (INIS)

    Highlights: → Acidification of autophagosome was blunted in steatotic hepatocytes. → Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. → Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. → Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  19. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

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    Altmeyer Peter

    2002-08-01

    Full Text Available Abstract Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2 has achieved great value within the treatment of severe atopic dermatitis (AD. The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with AD, biopsy specimens from 15 patients before and after treatment were analyzed immunohistochemically for proteolytic activation. Results Compared to lesional skin of patients with AD before UVA1 irradiation, the number of cells positive for cathepsin G within the dermal infiltrate decreased significantly after treatment. The decrease of cathepsin G+ cells was closely linked to a substantial clinical improvement in skin condition. Conclusions In summary, our findings demonstrated that medium-dose UVA1 irradiation leads to a modulation of the expression of cathepsin G in the dermal inflammatory infiltrate in patients with severe AD. Cathepsin G may attack laminin, proteoglycans, collagen I and insoluble fibronectin, to provoke proinflammatory events, to degrade the basement membrane, to destroy the tissue inhibitor of metalloproteinases and to increase the endothelial permeability. Therefore, its down-regulation by UVA1 phototherapy may induce the reduction of skin inflammation as well as improvement of the skin condition.

  20. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    Energy Technology Data Exchange (ETDEWEB)

    Inami, Yoshihiro [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Tanida, Isei [Department of Biochemistry and Cell Biology, Laboratory of Biomembranes, National Institute of Infectious Disease, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640 (Japan); Ikejima, Kenichi; Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  1. Prevalence and clinical significance of cathepsin G antibodies in systemic sclerosis

    Directory of Open Access Journals (Sweden)

    M. Favaro

    2011-09-01

    Full Text Available Objectives: To evaluate the prevalence and clinical significance of cathepsin G antibodies in patients affected with systemic sclerosis (SSc, scleroderma. Methods: 115 patients affected by SSc, 55 (47,8% with diffuse scleroderma (dSSc and 60 (52,2% with limited scleroderma (lSSc, were tested for cathepsin G antibodies by ELISA method. Moreover these sera were evaluated by indirect immunofluorescence (IIF on ethanol and formalin fixed human neutrophils. Results: By means of the ELISA method 16 (13,9% patients were found to be sera positive for anti-cathepsin G, 2 (12.5% of which showed a perinuclear fluorescence pattern (P-ANCA and 4 (25% an atypical ANCA staining, while 10 (62,5% were negative on IIF. The IIF on scleroderma sera revealed 5 (4,3% P-ANCA and 18 (15,7% atypical ANCA patterns. The anti-cathepsin G antibodies significantly prevailed in scleroderma sera (p=0.02 when their frequency was compared with that of healthy controls; while they were not significantly associated to any clinical or serological features of SSc patients. Conclusions: The anti-cathepsin G antibodies were significantly frequent in scleroderma sera; however, no clinical correlations were found. Thus, the significance of their presence in SSc still needs to be clarified.

  2. Leishmania aethiopica: identification and characterization of cathepsin L-like cysteine protease genes.

    Science.gov (United States)

    Kuru, Teklu; Jirata, Dagim; Genetu, Abebe; Barr, Stephen; Mengistu, Yohannes; Aseffa, Abraham; Gedamu, Lashitew

    2007-03-01

    There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis. PMID:17083936

  3. [Reducing bone resorption by cathepsin K inhibitor and treatment of osteoporosis].

    Science.gov (United States)

    Watanabe, Reiko; Okazaki, Ryo

    2014-01-01

    Cathepsin K is a lysosomal cysteine protease, secreted from osteoclasts. It plays a major role in the osteoclastic bone resorption by cleaving type 1 collagen, the major bone matrix protein, under acidic pH. In cathepsin K knockout mice, bone mineral density (BMD) is increased, bone resorption is decreased without reduction in the number of osteoclast whereas bone formation is decreased. Based on these results, cathepsin K inhibitors have been developed for the treatment of osteoporosis. Odanacatib is one of them and is perhaps closest for launching. In phase 1 and 2 trials, it markedly reduced bone resorption with a transient reduction in bone formation, thus resulted in a robust increase in both trabecular and cortical BMD in osteoporotics. Currently, Odanacatib is in phase 3 fracture prevention trial, of which results are anticipated in 2014. PMID:24369281

  4. Immunohistochemical localization of cathepsin D and a possible role of melanocytes during tail resorption in tadpoles of a tropical toad.

    Science.gov (United States)

    Mahapatra, Cuckoo; Mahapatra, Pravati Kumari

    2012-07-01

    Programmed cell death during anuran tail resorption is primarily brought about by apoptosis. Cathepsin D, a lysosomal aspartyl protease, is involved in the death of tail tissues. Thus, anuran tail resorption presents an ideal model to study cathepsin-mediated cell death during vertebrate development. Present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D in the tail tissues of the common Asian toad, Duttaphrynus melanostictus. Cathepsin D was involved in programmed cell death in epidermis, muscle, spinal cord, and blood cells in the resorbing tail. Interestingly, it was also involved in the pre-resorbing tail before visible tail resorption which indicates initiation of cell death even before actually the tail resorbs. Melanocytes were found to be one of the causative agents in degrading tail tissues and were associated with the degradation of muscle, epidermis and spinal cord of the resorbing tail. PMID:22505219

  5. Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus).

    Science.gov (United States)

    Liu, Yu-Xin; Zhou, Da-Yong; Ma, Dong-Dong; Liu, Yan-Fei; Li, Dong-Mei; Dong, Xiu-Ping; Tan, Ming-Qian; Du, Ming; Zhu, Bei-Wei

    2016-12-01

    The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. PMID:27374541

  6. Increased Circulating Cathepsin K in Patients with Chronic Heart Failure.

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    Guangxian Zhao

    Full Text Available Cysteinyl cathepsin K (CatK is one of the most potent mammalian collagenases involved in cardiovascular disease. Here, we investigated the clinical predictive value of serum CatK levels in patients with chronic heart failure (CHF. We examined 134 patients with CHF, measuring their serum CatK, troponin I, high-sensitive C-reactive protein, and pre-operative N-terminal pro-brain natriuretic peptide levels. The patients were divided into two groups: the 44 patients who showed a left ventricular (LV ejection fraction (LVEF < 40% (the "lowLVEF" group and the 90 patients showing LVEF values ≥ 40% (the "highLVEF" group. The lowLVEF patients had significantly higher serum CatK levels compared to the highLVEF patients (58.4 ± 12.2 vs. 44.7 ± 16.4, P < 0.001. Overall, a linear regression analysis showed that CatK levels correlated negatively with LVEF (r = -0.4, P < 0.001 and positively with LV end-diastolic dimensions (r = 0.2, P < 0.01, LV end-systolic dimensions (r = 0.3, P < 0.001, and left atrial diameters (r = 0.3, P < 0.01. A multiple logistic regression analysis showed that CatK levels were independent predictors of CHF (odds ratio, 0.90; 95% confidence interval, 0.84-0.95; P < 0.01. These data indicate that elevated levels of CatK are closely associated with the presence of CHF and that the measurement of circulating CatK provides a noninvasive method of documenting and monitoring the extent of cardiac remodeling and dysfunction in patients with CHF.

  7. Impact of cathepsin B on the interstitial fluid proteome of murine breast cancers.

    Science.gov (United States)

    Gomez-Auli, Alejandro; Hillebrand, Larissa Elisabeth; Biniossek, Martin Lothar; Peters, Christoph; Reinheckel, Thomas; Schilling, Oliver

    2016-03-01

    Carcinomas establish a molecular cross talk between malignant tumor cells and the activated non-malignant cells of the tumor stroma. This cell-cell communication in tumor-stroma interaction includes soluble, secreted proteins that act in a paracrine or autocrine manner. Proteases are crucial factors in tumor-stroma interaction by degrading or truncating secreted bioactive proteins. The cysteine protease cathepsin B is frequently overexpressed in several cancer types, including breast cancer. Its abundance often correlates with poor prognosis. In the murine polyoma virus middle T oncogene (PyMT) breast cancer model, cathepsin B is equally pro-tumorigenic. In this study, we investigate how cathepsin B shapes the secreted proteome of PyMT breast cancers. We employed a novel strategy to harvest tumor interstitial fluid (IF) in combination with chemical stable isotope tagging for quantitative proteomic comparison of IF stemming from PyMT tumors from wild-type mice, mice lacking cathepsin B, and mice over-expressing human cathepsin B. In three biological replicates, we achieve good proteome coverage (∼1700 proteins), with a large content (>70%) of secreted proteins. This characterizes IF as a robust source for the investigation of cancer secretomes. We also identified a large number of shed ectodomains, thus highlighting the importance of tumor-contextual cell surface proteolysis. Furthermore, IF contained >190 proteases and protease inhibitors, which span the entire range of absolute protein abundances; an observation testifying for an important role of proteolysis in tumor-stroma interaction. The cathepsin B genotype consistently affected proteins including alpha-1B-glycoprotein and major urinary proteins 11 and 8 (MUP8). Our study establishes tumor IF as a rich source for the investigation of secreted proteins in tumor biology and sheds light on complex proteolytic networks in the breast cancer secretome. PMID:26455267

  8. CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B.

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    Hiroaki Yoshii

    Full Text Available During a comparison of the infectivity of mNDK, a CD4-independent human immunodeficiency virus type 1 (HIV-1 strain, to various cell lines, we found that HeLa cells were much less susceptible than 293T and TE671 cells. Hybridoma cells between HeLa and 293T cells were as susceptible as 293T cells, suggesting that cellular factors enhance the mNDK infection in 293T cells. By screening a cDNA expression library in HeLa cells, cystatin C was isolated as an enhancer of the mNDK infection. Because cathepsin B protease, a natural ligand of cystatin C, was upregulated in HeLa cells, we speculated that the high levels of cathepsin B activities were inhibitory to the CD4-independent infection and that cystatin C enhanced the infection by impairing the excessive cathepsin B activity. Consistent with this idea, pretreatment of HeLa cells with 125 µM of CA-074Me, a cathepsin B inhibitor, resulted in an 8-fold enhancement of the mNDK infectivity. Because cathepsin B is activated by low pH in acidic endosomes, we further examined the potential roles of endosomes in the CD4-independent infection. Suppression of endosome acidification or endocytosis by inhibitors or by an Eps15 dominant negative mutant reduced the infectivity of mNDK in which CD4-dependent infections were not significantly impaired. Taken together, these results suggest that endocytosis, endosomal acidification, and cathepsin B activity are involved in the CD4-independent entry of HIV-1.

  9. Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism

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    Flynn Claudia T

    2011-07-01

    Full Text Available Abstract Background In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease. Results Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. In vitro overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis. Conclusions Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our in vitro studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.

  10. Cathepsin Gene Family Reveals Transcriptome Patterns Related to the Infective Stages of the Salmon Louse Caligus rogercresseyi.

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    Waleska Maldonado-Aguayo

    Full Text Available Cathepsins are proteases involved in the ability of parasites to overcome and/or modulate host defenses so as to complete their own lifecycle. However, the mechanisms underlying this ability of cathepsins are still poorly understood. One excellent model for identifying and exploring the molecular functions of cathepsins is the marine ectoparasitic copepod Caligus rogercresseyi that currently affects the Chilean salmon industry. Using high-throughput transcriptome sequencing, 56 cathepsin-like sequences were found distributed in five cysteine protease groups (B, F, L, Z, and S as well as in an aspartic protease group (D. Ontogenic transcriptome analysis evidenced that L cathepsins were the most abundant during the lifecycle, while cathepsins B and K were mostly expressed in the larval stages and adult females, thus suggesting participation in the molting processes and embryonic development, respectively. Interestingly, a variety of cathepsins from groups Z, L, D, B, K, and S were upregulated in the infective stage of copepodid, corroborating the complexity of the processes involved in the parasitic success of this copepod. Putative functional roles of cathepsins were conjectured based on the differential expressions found and on roles previously described in other phylogenetically related species. Moreover, 140 single nucleotide polymorphisms (SNP were identified in transcripts annotated for cysteine and aspartic proteases located into untranslated regions, or the coding region. This study reports for the first time the presence of cathepsin-like genes and differential expressions throughout a copepod lifecycle. The identification of cathepsins together with functional validations represents a valuable strategy for pinpointing target molecules that could be used in the development of new delousing drugs or vaccines against C. rogercresseyi.

  11. Role of endocytosis and cathepsin-mediated activation in Nipah virus entry

    International Nuclear Information System (INIS)

    The recent discovery that the Nipah virus (NiV) fusion protein (F) is activated by endosomal cathepsin L raised the question if NiV utilize pH- and protease-dependent mechanisms of entry. We show here that the NiV receptor ephrin B2, virus-like particles and infectious NiV are internalized from the cell surface. However, endocytosis, acidic pH and cathepsin-mediated cleavage are not necessary for the initiation of infection of new host cells. Our data clearly demonstrate that proteolytic activation of the NiV F protein is required before incorporation into budding virions but not after virus entry

  12. Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages

    Science.gov (United States)

    Lah, T. T.; Hawley, M.; Rock, K. L.; Goldberg, A. L.

    1995-01-01

    Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

  13. Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori

    International Nuclear Information System (INIS)

    The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed

  14. Molecular modeling approach to explore the role of cathepsin B from Hordeum vulgare in the degradation of Aβ peptides.

    Science.gov (United States)

    Dhanavade, Maruti J; Parulekar, Rishikesh S; Kamble, Subodh A; Sonawane, Kailas D

    2016-01-01

    The pathological hallmark of Alzheimer's disease is the accumulation of Aβ peptides in human brains. These Aβ peptides can be degraded by several enzymes such as hACE, hECE, hIDE and cathepsin B. Out of which cathepsin B also belongs to the papain super family and has been found in human brains, it has a role in Aβ peptide degradation through limited proteolysis. The Aβ concentrations are maintained properly by its production and clearance via receptor-mediated cellular uptake and direct enzymatic degradation. However, the reduced production of Aβ degrading enzymes as well as their Aβ degrading activity in human brains initiate the process of accumulation of Aβ peptides. So it becomes essential to investigate the molecular interactions involved in the process of Aβ degradation in detail at the atomic level. Hence, homology modeling, molecular docking and molecular dynamics simulation techniques have been used to explore the possible role of cathepsin B from Hordeum vulgare in the degradation of amyloid beta (Aβ) peptides. The homology model of cathepsin B from Hordeum vulgare shows good similarity with human cathepsin B. Molecular docking and MD simulation results revealed that the active site residues Cys32, HIS112, HIS113 are involved in the catalytic activity of cathepsin B. The sulfhydryl group of the Cys32 residue of cathepsin B from Hordeum vulgare cleaves the Aβ peptide from the carboxylic end of Glu11. Hence, this structural study might be helpful in designing alternative strategies for the treatment of AD. PMID:26568474

  15. Prognostic and predictive value of cathepsin X in serum from colorectal cancer patients

    DEFF Research Database (Denmark)

    Vižin, Tjaša; Christensen, Ib Jarle; Wilhelmsen, Michael; Nielsen, Hans Jørgen; Kos, Janko

    2014-01-01

    BACKGROUND: Cathepsin X is a cysteine protease involved in mechanisms of malignant progression. It is secreted from tumour cells as a proenzyme and may serve to predict the disease status and risk of death for cancer patients. In a previous, pilot, study on 77 colorectal patients we demonstrated...

  16. Purification and characterization of cathepsin D from herring muscle ( Clupea harengus )

    DEFF Research Database (Denmark)

    Nielsen, L.B.; Nielsen, Henrik Hauch

    2001-01-01

    Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38 000-39 000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at ...

  17. Cathepsin D SNP associated with increased risk of variant Creutzfeldt-Jakob disease

    Directory of Open Access Journals (Sweden)

    Sanchez-Juan Pascual

    2008-04-01

    Full Text Available Abstract Background Variant Creutzfeldt-Jakob disease (vCJD originally resulted from the consumption of foodstuffs contaminated by bovine spongiform encephalopathy (BSE material, with 163 confirmed cases in the UK to date. Many thousands are likely to have been exposed to dietary infection and so it is important (for surveillance, epidemic modelling, public health and understanding pathogenesis to identify genetic factors that may affect individual susceptibility to infection. This study looked at a polymorphism in the cathepsin D gene (refSNP ID: rs17571 previously examined in Alzheimer's disease (AD. Methods Blood samples taken from 110 vCJD patients were tested for the C-T base change, and genotype data were compared with published frequencies for a control population using multiple logistic regression. Results There was a significant excess of the cathepsin D polymorphism TT genotype in the vCJD cohort compared to controls. The TT genotype was found to have a 9.75 fold increase in risk of vCJD compared to the CT genotype and a 10.92 fold increase compared to the CC genotype. Conclusion This mutation event has been observed to alter the protease activity of the cathepsin D protein and has been linked to an increase in amyloid beta plaque formation in AD. vCJD neuropathology is characterised by the presence of amyloid plaques, formed from the prion protein, and therefore alterations in the amyloid processing activity of cathepsin D may affect the neuropathogenesis of this disease.

  18. Genetic and pharmacological evidence implicates cathepsins in Niemann-Pick C cerebellar degeneration.

    Science.gov (United States)

    Chung, Chan; Puthanveetil, Prasanth; Ory, Daniel S; Lieberman, Andrew P

    2016-04-01

    Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease. PMID:26908626

  19. Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth.

    Directory of Open Access Journals (Sweden)

    Elias Gounaris

    Full Text Available It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4 of the probe signal was localized in CD11b(+Gr1(+ myeloid derived suppressor cells (MDSC and CD11b(+F4/80(+ macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.

  20. Molecular Cloning and Sequence of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Cathepsin S gene

    Science.gov (United States)

    Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. This enzyme digests the invariant chain molecules so that antigenic peptides are able to load on the class II-associated invariant chain peptide of MHC. The complexes can subsequently be presented to the CD4 cell surface. In ...

  1. Activation route of the Schistosoma mansoni cathepsin B1 drug target

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Horn, Martin; Řezáčová, Pavlína; Marešová, Lucie; Fajtová, Pavla; Brynda, Jiří; Vondrášek, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 39. ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : Schistosoma mansoni * cathepsin B1 * sulfated polysaccharides Subject RIV: CE - Biochemistry

  2. Cathepsin B protease is required for metamorphism in silkworm,Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Gen-Hong Wang; Chun Liu; Qing-You Xia; Xing-Fu Zha; Jie Chen; Liang Jiang

    2008-01-01

    Cathepsin B belongs to lysosomal cysteine protease of the papain family.Temporal and spatial expression analysis of cathepsin B of Bombyx mori (BmCtB) was carried out based on Expression Sequence Tags (ESTs) data,oligonucleotide microarray,reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR.Expression of BmCtB was observed in all of the tissues and stages.Among the 10 tested tissues,the fat body and posterior silk gland are the two most enriched tissues with BmCtB.During Bombyx development,there was an expression fastigium of BmCtB during metamorphosis.RNA interference was used to suppress the expression of cathepsin B during metamorphosis.Significant developmental defective phenotypes were obtained in the RNAi treated group.The dramatically reduced expression of BmCtB was confirmed by Northern blot and quantitative real-time PCR.These evidences strongly suggest cathepsin B proteinase was predominantly involved in the metabolism process of fat body and the posterior silk gland and was critical for metamorphism and development of silkworm,Bombyx mori.

  3. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

    Science.gov (United States)

    de Castro, M A G; Bunt, G; Wouters, F S

    2016-01-01

    The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis. PMID:27551506

  4. Caffeine suppresses the progression of human glioblastoma via cathepsin B and MAPK signaling pathway.

    Science.gov (United States)

    Cheng, Yu-Chen; Ding, You-Ming; Hueng, Dueng-Yuan; Chen, Jang-Yi; Chen, Ying

    2016-07-01

    Glioblastoma has aggressive proliferative and invasive properties. We investigated the effect of caffeine on the invasion and the anti-cancer effect in human glioblastomas. Caffeine reduced the invasion in U-87MG, GBM8401 and LN229 cells. Caffeine decreased mRNA, protein expression, and activity of cathepsin B. Besides, mRNA and protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) was upregulated by caffeine treatment, whereas matrix metalloproteinase-2 (MMP-2) was downregulated. The expression of Ki67, p-p38, phospforylated extracellular regulated protein kinases (p-ERK), and membranous integrin β1 and β3 was decreased by caffeine. The Rho-associated protein kinase (ROCK) inhibitor, Y27632, blocked the caffeine-mediated reduction of cathepsin B, phosphorylated focal adhesion kinase (p-FAK), and p-ERK, and invasion. Moreover, caffeine decreased the tumor size, cathepsin B and Ki67 expression in animal model. Caffeine reduced the invasion of glioma cells through ROCK-cathepsin B/FAK/ERK signaling pathway and tumor growth in orthotopic xenograft animal model, supporting the anti-cancer potential in glioma therapy. PMID:27260469

  5. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Roč. 90, č. 3 (2008), s. 349-357. ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

  6. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    Energy Technology Data Exchange (ETDEWEB)

    Schreuder, Herman A., E-mail: herman.schreuder@sanofi.com; Liesum, Alexander, E-mail: alexander.liesum@sanofi.com; Kroll, Katja, E-mail: katja.kroll@sanofi.com; Böhnisch, Britta, E-mail: britta.boehnisch@sanofi.com; Buning, Christian, E-mail: christian.buning@sanofi.com; Ruf, Sven, E-mail: sven.ruf@sanofi.com; Sadowski, Thorsten, E-mail: thorsten.sadowski@sanofi.com

    2014-03-07

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  7. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    International Nuclear Information System (INIS)

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  8. Cathepsin B and uPAR Knockdown Inhibits Tumor-induced Angiogenesis by Modulating VEGF Expression in Glioma

    OpenAIRE

    MALLA, RAMA RAO; Gopinath, Sreelatha; Christopher S Gondi; Alapati, Kiranmai; Dinh, Dzung H.; Gujrati, Meena; Rao, Jasti S.

    2011-01-01

    Angiogenesis, which is the process of sprouting of new blood vessels from pre-existing vessels, is vital for tumor progression. Proteolytic remodeling of extracellular matrix is a key event in vessel sprouting during angiogenesis. Urokinase plasminogen activator receptor (uPAR) and cathepsin B are both known to be overexpressed and implicated in tumor angiogenesis. In the present study, we observed that knockdown of uPAR and cathepsin B using puPAR (pU), pCathepsin B (pC), and a bicistronic c...

  9. Redox-based inactivation of cysteine cathepsins by compounds containing the 4-aminophenol moiety.

    Directory of Open Access Journals (Sweden)

    Bojana Mirković

    Full Text Available BACKGROUND: Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming. METHODS AND FINDINGS: In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a k(inact/K(I of 36.7 ± 13.6 M(-1 s(-1 using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with k(inact/K(I values of 406.4 ± 10.8 and 36.5 ± 1.3 M(-1 s(-1. No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS by 4-aminophenols in various redox environments was determined by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H(2O(2 is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid. CONCLUSIONS: In this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases.

  10. Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo.

    Science.gov (United States)

    Vermeire, Jon J; Suzuki, Brian M; Caffrey, Conor R

    2016-01-01

    Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite's resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck's cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite's CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN's somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole. PMID:27384569

  11. Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo

    Directory of Open Access Journals (Sweden)

    Jon J. Vermeire

    2016-07-01

    Full Text Available Hookworm infection is chief among soil-transmitted helminthiases (STHs for the chronic morbidly inflicted. Deworming via mass drug administration (MDA programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN, Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole.

  12. Cathepsin D gene Expression in Stomach: Its Association with Age, Sex, and Menopausal status

    Directory of Open Access Journals (Sweden)

    Reza Abedi

    2016-03-01

    Full Text Available Background & Objectives :Gastric cancer is 2-4 folds higher in men than women. Sex hormones are one of the leading causes of sexual dimorphism in incidence of gastric cancer. The aim of this study is to compare Cathepsin D and Caspase-7 gene expressions in the gastric tissue of normal men and women. Materials & Methods :In this cross-sectional study, gastric antrum tissue samples were collected from 21 healthy females and 21 males in the three age groups including 35, 35-50, and over 50 years. Following RNA extraction and cDNA synthesis, the expressions of genes were compared between men and women via semi-quantitative Reverse Transcription-PCR method. The obtained data were analyzed, using the statistical T-Test and ANOVA. Results: Statical analyses confirmed that the expression of Cathepsin D gene was significantly higher in men under 35 than those in the range of 35-50 years (p=0.04. In addition, the expression of Cathepsin D gene was significantly 10 folds in pre-menopause than post-menopause women and men (post-menopause women and men as one group (p=0.008. Furthermore, the expression of Cathepsin D gene between men and women was significant at borderline (p=0.056. Conclusion: The findings of the present research indicate that the expression of Cathepsin D is higher in pre-menopause than post-menopause women and men, and is greater in men under 35 than those in the range of 35-50 years.

  13. Decreased expression of APAF-1 and increased expression of cathepsin B in invasive pituitary adenoma

    Directory of Open Access Journals (Sweden)

    Tanase C

    2014-12-01

    Full Text Available Cristiana Tanase,1 Radu Albulescu,1,2 Elena Codrici,1 Bogdan Calenic,1,3 Ionela Daniela Popescu,1 Simona Mihai,1 Laura Necula,1,4 Maria Linda Cruceru,5 Mihail Eugen Hinescu1,5 1“Victor Babes” National Institute of Pathology, Biochemistry-Proteomics Department, 2National Institute for Chemical Pharmaceutical R&D, 3“Carol Davila” University of Medicine and Pharmacy, Department of Biochemistry, 4Stefan S. Nicolau Institute of Virology, Cellular and Molecular Pathology, 5“Carol Davila” University of Medicine and Pharmacy, Cellular and Molecular Medicine Department, Bucharest, Romania Purpose: Apoptotic protease-activating factor-1 (APAF-1 and cathepsin B are important functional proteins in apoptosis; the former is involved in the intrinsic (mitochondrial pathway, while the latter is associated with both intrinsic and extrinsic pathways. Changes in the expression of apoptosome-related proteins could be useful indicators of tumor development since a priori defects in the mitochondrial pathway might facilitate the inception and progression of human neoplasms. Our aim was to evaluate the profiles of APAF-1 and cathepsin B in relation with other molecules involved in apoptosis/proliferation and to correlate them with the aggressive behavior of invasive pituitary adenomas. Materials and methods: APAF-1 and cathepsin B were assessed in tissue samples from 30 patients with pituitary adenomas, of which 16 were functional adenomas and 22 were invasive adenomas. Results: A positive relationship between high proliferation and invasiveness was observed in invasive pituitary adenomas when compared to their noninvasive counterparts (Ki-67 labeling index – 4.72% versus 1.75%. Decreased expression of APAF-1 was recorded in most of the invasive adenomas with a high proliferation index, while the cathepsin B level was elevated in this group. We have noticed a negative correlation between the low level of APAF-1 and invasiveness (63.63%; P<0.01; at the

  14. Study of Low-grade Chronic Inflammatory Markers in Men with Central Obesity: Cathepsin S was Correlated with Waist Circumference

    Directory of Open Access Journals (Sweden)

    Adriana Todingrante

    2013-08-01

    Full Text Available BACKGROUND: There is a prevalence increase of overweight and obesity in Indonesia. Central obesity can lead a variety of chronic diseases through the inflammatory process. There are some markers for low-grade chronic inflammatory, such as cathepsin S, high sensitivity C-reactive protein (hs-CRP, interleukin-1- beta (IL-1β. To our current interest that central obesity can lead to various chronic diseases through the inflammatory process, we conducted a study to investigate correlation of Cathepsin S, hs-CRP, IL-1β in men with central obesity. METHODS: A cross-sectional study was conducted. Seventy-eight selected subjects were examined to collect anthropometric data and prepared for sample collection. Collected samples were processed for the following biochemical analyses: fasting glucose, high density lipoprotein (HDL-cholesterol, triglyceride, serum glutamic oxaloacetic transaminase (SGOT, serum glutamate pyruvate transaminase (SGPT, cathepsin S, hs-CRP, and IL-1β. Data distribution and variable correlation were then statistically analyzed. RESULTS: There were significant correlations between waist circumference (WC and cathepsin S (p=0.030; r=0.214, hs-CRP and cathepsin S (p=0.007; r=0.276, triglyceride and IL-1β (p=0.019; r=-0.235, WC and systolic blood pressure (SBP (p=0.003; r=-0.312, WC and fasting glucose (p=0.000; r=0.380, WC and body mass index (BMI (p=0.000; r=0.708. CONCLUSIONS: Our study showed that cathepsin S was correlated with central obesity, suggesting that cathepsin S could be a potential inflammatory marker in central obesity in the future. KEYWORDS: obesity, inflammation, hs-CRP, cathepsin S, IL-1β, waist circumference.

  15. Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design.

    OpenAIRE

    Baldwin, E. T.; Bhat, T N; Gulnik, S; Hosur, M. V.; Sowder, R C; Cachau, R.E.; Collins, J.; A. M. Silva; Erickson, J. W.

    1993-01-01

    Cathepsin D (EC 3.4.23.5) is a lysosomal protease suspected to play important roles in protein catabolism, antigen processing, degenerative diseases, and breast cancer progression. Determination of the crystal structures of cathepsin D and a complex with pepstatin at 2.5 A resolution provides insights into inhibitor binding and lysosomal targeting for this two-chain, N-glycosylated aspartic protease. Comparison with the structures of a complex of pepstatin bound to rhizopuspepsin and with a h...

  16. Proteomic Analysis of Circulating Monocytes Identifies Cathepsin D as A Potential Novel Plasma Marker of Acute Coronary Syndromes

    Directory of Open Access Journals (Sweden)

    Fernando Vivanco

    2008-01-01

    Full Text Available We have performed a proteomic analysis of peripheral blood monocytes from ACS patients in comparison with healthy subjects and stable coronary patients in order to search novel biomarkers of ACS in circulating monocytes. Monocytes were isolated from blood of patients with non-ST elevation ACS (n = 27 at day 0, 2 and 6 months, and from patients with stable coronary disease (n = 10 and matched healthy controls (n = 11. The proteomic analysis of monocytes from ACS patients at day 0 showed that cathepsin D is differentially expressed compared to healthy subjects and stable coronary patients. Western blot analysis indicated that the mature form of cathepsin D at day 0 was overexpressed in monocytes of ACS patients in relation to healthy subjects. In contrast, the precursor of this enzyme, absent at day 0 in ACS patients, was highly expressed in monocytes of healthy subjects. Furthermore, the upregulation of the mature form of cathepsin D diminished along the time, while the expression of the precursor increased. ACS patients also showed significantly increased plasma cathepsin D levels on admission compared to healthy subjects and stable patients. Cathepsin D plasma levels diminished at 2 and 6 months to control values. Finally, cathepsin D levels were independent of the existence of coronary risk factors and CRP levels, correlating only with CD40L. Since this protease participates in the genesis and rupture of atherosclerotic plaques, it could represent a potential marker of ACS.

  17. Immunocapture of circulating Schistosoma mansoni cathepsin B antigen (Sm31) by anti-Sm31 polyclonal antibodies.

    Science.gov (United States)

    González, Amelia Y; Sulbarán, Guidenn S; Ballen, Diana E; Cesari, Italo M

    2016-06-01

    Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms. Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients. Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA). To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography. Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr.~31kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis. CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections. PMID:26709076

  18. Strategies for detection and quantification of cysteine cathepsins-evolution from bench to bedside.

    Science.gov (United States)

    Hughes, Caroline S; Burden, Roberta E; Gilmore, Brendan F; Scott, Christopher J

    2016-03-01

    The cysteine cathepsins are a family of closely related thiol proteases, normally found in the endosomal and lysosomal compartments of cells. A growing body of evidence has clearly linked the dysregulated activity of these proteases with many diseases and pathological conditions, offering therapeutic, prognostic and diagnostic potential. However, these proteases are synthesised as inactive precursors and once activated, are controlled by factors such as pH and presence of endogenous inhibitors, meaning that overall protein and activity levels do not necessarily correlate. In order to fully appreciate the role and potential of these proteases, tools are required that can detect and quantify overall cathepsin activity. Two main strategies have evolved; synthetic substrates and protease-labelling with affinity-binding probes (or activity-based probes). This review examines recent innovations in these approaches as the field moves towards developing tools that could ultimately be used in patients for diagnostic or prognostic applications. PMID:26253694

  19. Human macrophage foam cells degrade atherosclerotic plaques through cathepsin K mediated processes

    DEFF Research Database (Denmark)

    Barascuk, Natasha; Skjøt-Arkil, Helene; Register, Thomas C; Larsen, Lise; Byrjalsen, Inger; Christiansen, Claus; Karsdal, Morten A

    2010-01-01

    BACKGROUND: Proteolytic degradation of Type I Collagen by proteases may play an important role in remodeling of atherosclerotic plaques, contributing to increased risk of plaque rupture.The aim of the current study was to investigate whether human macrophage foam cells degrade the extracellular...... matrix (ECM) of atherosclerotic plaques by cathepsin K mediated processes. METHODS: We 1) cultured human macrophages on ECM and measured cathepsin K generated fragments of type I collagen (C-terminal fragments of Type I collagen (CTX-I) 2) investigated the presence of CTX-I in human coronary arteries and......-I in areas of intimal hyperplasia and in shoulder regions of advanced plaques. Treatment of human monocytes with M-CSF or M-CSF+LDL generated macrophages and foam cells producing CTX-I when cultured on type I collagen enriched matrix. Circulating levels of CTX-I were not significantly different in...

  20. Optical Imaging of Cancer-Related Proteases Using Near-Infrared Fluorescence Matrix Metalloproteinase-Sensitive and Cathepsin B-Sensitive Probes

    OpenAIRE

    Yhee, Ji Young; Kim, Sun Ah; Koo, Heebeom; Son, Sohee; Ryu, Ju Hee; Youn, In-Chan; Choi, Kuiwon; Kwon, Ick Chan; Kim, Kwangmeyung

    2012-01-01

    Cathepsin B and matrix metalloproteinase (MMP) play key roles in tumor progression by controlled degradation of extracellular matrix. Consequently, these proteases have been attracted in cancer research, and many imaging probes utilizing these proteases have been developed. Our groups developed cathepsin B and MMP imaging nanoprobes based on polymer nanoparticle platform. Both cathepsin B and MMP imaging probes used near-infrared fluorescence (NIRF) dye and dark-quencher to for high sensitivi...

  1. Utility of a Protein Fraction with Cathepsin L-Like Activity Purified from Cysticercus Fluid of Taenia solium in the Diagnosis of Human Cysticercosis

    OpenAIRE

    Zimic, Mirko; Pajuelo, Mónica; Rueda, Daniel; López, César; Arana, Yanina; Castillo, Yesenia; Calderón, Maritza; Rodriguez, Silvia; Sheen, Patricia; Vinetz, Joseph M.; Gonzales, Armando; García, Héctor H.; Gilman, Robert H.

    2009-01-01

    Neurocysticercosis, an endemic parasitic disease in most developing countries, is caused by Taenia solium and compromises the human central nervous system. Cathepsin L-like proteases are secreted by several parasites including T. solium and constitute important antigens for immunodiagnostics. A protein fraction with cathepsin L-like activity was purified from the cysticercus fluid by size exclusion and ion exchange chromatography. Cathepsin L-like activity was measured fluorometrically by det...

  2. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    OpenAIRE

    Marton Siklos; Manel BenAissa; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families...

  3. Gene targeting of the cysteine peptidase cathepsin H impairs lung surfactant in mice.

    Directory of Open Access Journals (Sweden)

    Frank Bühling

    Full Text Available BACKGROUND: The 11 human cysteine cathepsins are proteases mainly located in the endolysosomal compartment of all cells and within the exocytosis pathways of some secretory cell types. Cathepsin H (Ctsh has amino- and endopeptidase activities. In vitro studies have demonstrated Ctsh involvement in the processing and secretion of the pulmonary surfactant protein B (SP-B. Furthermore, Ctsh is highly expressed in the secretory organelles of alveolar type II pneumocytes where the surfactant proteins are processed. METHODOLOGY/PRINCIPAL FINDINGS: Hence, we generated Ctsh null mice by gene targeting in embryonic stem cells to investigate the role of this protease in surfactant processing in vivo. The targeting construct contains a ß-galactosidase (lacZ reporter enabling the visualisation of Ctsh expression sites. Ctsh-deficiency was verified by northern blot, western blot, and measurement of the Ctsh aminopeptidase activity. Ctsh(-/- mice show no gross phenotype and their development is normal without growth retardation. Broncho-alveolar lavage (BAL from Ctsh(-/- mice contained lower levels of SP-B indicating reduced SP-B secretion. The BAL phospholipid concentration was not different in Ctsh(+/+ and Ctsh(-/- mice, but measurement of surface tension by pulsating bubble surfactometry revealed an impairment of the tension reducing function of lung surfactant of Ctsh(-/- mice. CONCLUSIONS/SIGNIFICANCE: We conclude that cathepsin H is involved in the SP-B production and reduced SP-B levels impair the physical properties of the lung surfactant. However, Ctsh defiency does not reproduce the severe phenotype of SP-B deficient mice. Hence, other proteases of the secretory pathway of type II pneumocytes, i.e. cathepsins C or E, are still able to produce surfactant of sufficient quality in absence of Ctsh.

  4. Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser

    OpenAIRE

    L. Redecke; Nass, K.; DePonte, D. P.; White, T A; Rehders, D.; Barty, A.; F. Stellato; Liang, M; Barends, T. R. M.; Boutet, S.; Williams, G J; Messerschmidt, M.; Seibert, M. M.; Aquila, A.; Arnlund, D.

    2012-01-01

    The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, ...

  5. Impact of cathepsins on the activation of proinsulin-reactive T cells in type 1 diabetes

    OpenAIRE

    Zou, Fang

    2012-01-01

    Autoantigenic peptides resulting from self-proteins such as proinsulin are important players in the development of type 1 diabetes mellitus (T1D). Self-proteins can be processed by cathepsins (Cats) within endocytic compartments and loaded to major histocompatibility complex (MHC) class II molecules for CD4+ T cell inspection. However, the processing and presentation of proinsulin by antigen-presenting cells (APC) in humans is only partially understood. Here we demonstrate that the processing...

  6. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

  7. Atick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

    Czech Academy of Sciences Publication Activity Database

    Chmelař, Jindřich; Oliveira, C. J.; Řezáčová, Pavlína; Francischetti, I.M.B.; Kovářová, Zuzana; Pejler, G.; Kopáček, Petr; Ribeiro, J.M.C.; Mareš, Michael; Kopecký, Jan; Kotsyfakis, Michalis

    2011-01-01

    Roč. 117, č. 2 (2011), s. 736-744. ISSN 0006-4971 R&D Projects: GA AV ČR IAA600960811; GA MŠk(CZ) LC06009; GA ČR(CZ) GAP207/10/2183 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : parasite serpin * IRS-2 * tick * Ixodes ricinus * platelet aggregation * inflammation * cathepsin G * chymase Subject RIV: EC - Immunology Impact factor: 9.898, year: 2011

  8. Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Řezáčová, Pavlína; Lepšík, Martin; Horn, Martin; Váchová, Jana; Fanfrlík, Jindřich; Brynda, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2011-01-01

    Roč. 286, č. 41 (2011), s. 35770-35881. ISSN 0021-9258 R&D Projects: GA ČR GA203/09/1585; GA ČR GAP208/11/0295; GA MŠk OC09007 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : cathepsin B * schistosoma * crystal structure Subject RIV: CC - Organic Chemistry Impact factor: 4.773, year: 2011

  9. CNS-Expressed Cathepsin D Prevents Lymphopenia in a Murine Model of Congenital Neuronal Ceroid Lipofuscinosis

    OpenAIRE

    Shevtsova, Zinayida; Garrido, Manuel; Weishaupt, Jochen; Saftig, Paul; Bähr, Mathias; Lühder, Fred; Kügler, Sebastian

    2010-01-01

    Deficiency in Cathepsin D (CtsD), the major cellular lysosomal aspartic proteinase, causes the congenital form of neuronal ceroid lipofuscinoses (NCLs). CtsD-deficient mice show severe visceral lesions like lymphopenia in addition to their central nervous system (CNS) phenotype of ceroid accumulation, microglia activation, and seizures. Here we demonstrate that re-expression of CtsD within the CNS but not re-expression of CtsD in visceral organs prevented both central and visceral pathologies...

  10. Novel Compound Heterozygous Mutations in the Cathepsin K Gene in Japanese Female Siblings with Pyknodysostosis

    OpenAIRE

    Matsushita, M.(CNS, The University of Tokyo, RIKEN Campus, Saitama, Wako, 351-0198, Japan); Kitoh, H.; Kaneko, H.; Mishima, K.; Itoh, Y.; Hattori, T; Ishiguro, N

    2012-01-01

    We report on female siblings with pyknodysostosis who showed common clinical and radiographic features including disproportionate short stature, dental abnormalities, increased bone density, open fontanelle, and acroosteolysis. Sequence analysis of the cathepsin K (CTSK) gene demonstrated compound heterozygous mutations (935 C>T, A277V and 489 G>C, R122P) in the affected siblings and a heterozygous mutation in their parents. The former missense mutation has previously been reported in 6 unrel...

  11. Cathepsins B1 and B2 of Trichobilharzia spp., bird schistosomes causing cercarial dermatitis

    Czech Academy of Sciences Publication Activity Database

    Kašný, M.; Mikeš, L.; Dolečková, K.; Hampl, V.; Dvořák, Jan; Novotný, M.; Horák, P.

    BERLIN: SPRINGER-VERLAG BERLIN, HEIDELBERGER PLATZ 3, D-14197, 2011 - (Robinson, M.; Dalton, J.), s. 136-154. (Advances in Experimental Medicine and Biology. 712). ISBN 978-1-4419-8413-5 Institutional research plan: CEZ:AV0Z60220518 Keywords : Trichobilharzia * trematode * Schistosoma * Fasciola * peptidase * cercarial elastase * cathepsin * hydrolysis * blood digestion * tissue migration * biochemical characterization * phylogeny * function Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine

  12. Cathepsin F Cysteine Protease of the Human Liver Fluke, Opisthorchis viverrini

    OpenAIRE

    Pinlaor, Porntip; Kaewpitoon, Natthawut; Laha, Thewarach; Sripa, Banchob; Kaewkes, Sasithorn; Morales, Maria E.; Mann, Victoria H.; Parriott, Sandi K.; Suttiprapa, Sutas; Robinson, Mark W.; To, Joyce; Dalton, John P.; Loukas, Alex; Brindley, Paul J.

    2009-01-01

    Background The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Methodology/Principal Findings Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocali...

  13. Cathepsin D precursors in clathrin-coated organelles from human fibroblasts

    OpenAIRE

    1985-01-01

    Coated vesicles were isolated from metabolically labeled human fibroblasts with the aid of affinity-purified antibodies against human brain clathrin and Staphylococcus aureus cells. The material adsorbed to the S. aureus cells was enriched in clathrin. When the S. aureus cells bearing the immunoadsorbed material were treated with 0.5% saponin, extracts containing the precursor form of cathepsin D were obtained. The extraction of the precursor was promoted in the presence of mannose 6-phosphat...

  14. Cathepsin K inhibitors increase distal femoral bone mineral density in rapidly growing rabbits

    OpenAIRE

    Pennypacker, Brenda L; Oballa, Renata M; Levesque, Sonia; Kimmel, Donald B.; Duong, Le T.

    2013-01-01

    Background Selective and reversible inhibitors of human Cathepsin K (CatK), including odanacatib (ODN), have been developed as potential therapeutics for the treatment of osteoporosis. Inhibitors of human CatK show significantly less potency for the rodent enzymes compared with that for the human or rabbit enzymes; thus the Schenk model in growing rabbit was developed as a screening assay for the in vivo activity of CatK inhibitors in blocking bone resorption. Methods In this study, the effic...

  15. Therapeutic inhibition of cathepsin K—reducing bone resorption while maintaining bone formation

    OpenAIRE

    Duong, Le T.

    2012-01-01

    Osteoporosis is a disease of high bone remodeling with an imbalance of bone resorption over bone formation, resulting in decreased bone mineral density and deterioration of bone microarchitecture. From the emerging understandings of the molecular and cellular regulators of bone remodeling, potential new targets for therapeutic intervention for this disease have been identified. Cathepsin K (CatK), a cysteine protease produced by osteoclasts, is the primary enzyme mediating the degradation of ...

  16. Overexpression of cathepsin f, matrix metalloproteinases 11 and 12 in cervical cancer

    International Nuclear Information System (INIS)

    Cervical carcinoma (CC) is one of the most common cancers among women worldwide and the first cause of death among the Mexican female population. CC progression shows a continuum of neoplastic transitions until invasion. Matrix metalloproteinases (MMPs) and cathepsins play a central role on the enhancement of tumor-induced angiogenesis, cell migration, proliferation, apoptosis and connective tissue degradation. MMPs -2 and -9 expression has been widely studied in cervical cancer. Nevertheless, no other metalloproteinases or cathepsins have been yet related with the progression and/or invasion of this type of cancer. Three HPV18 CC cell lines, two HPV16 CC cell lines and three HPV16 tumor CC tissues were compared with three morphologically normal, HPV negative, cervical specimens by cDNA arrays. Overexpression of selected genes was confirmed by end point semiquantitative reverse transcription-PCR with densitometry. In situ hybridization and protein expression of selected genes was further studied by means of two tissue microarrays, one consisting of 10 HSIL and 15 CC and the other one of 15 normal cervical and 10 LSIL tissues. TIMP1, Integrins alpha 1 and 4, cadherin 2 and 11, Cathepsins F, B L2, MMP 9, 10 11 and 12 were upregulated and Cathepsin S, L, H and C, Cadherins 3 and 4, TIMP3, MMP 13, Elastase 2 and Integrin beta 8 were found to be downregulated by cDNA arrays. Endpoint RT-PCR with densitometry gave consistent results with the cDNA array findings for all three genes selected for study (CTSF, MMP11 and MMP12). In situ hybridization of all three genes confirmed overexpression in all the HSIL and CC. Two of the selected proteins were detected in LSIL, HSIL and CC by immunohistochemistry. Novel undetected CC promoting genes have been identified. Increased transcription of these genes may result in overexpression of proteins, such as CTSF, MMP11 and MMP12 which could contribute to the pathogenesis of CC

  17. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation.

    Science.gov (United States)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-12-30

    Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings. PMID:26223015

  18. Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

    Science.gov (United States)

    Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

    2016-07-01

    Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

  19. Changes of malonaldehyde, cathepsin D and α2-macroglobulin (α2M) after ionizing radiation injury

    International Nuclear Information System (INIS)

    Increases in levels of malonaldehyde in plasma, liver and kidney, and increases of tissue kallikrein in kidney and urine were found in Wistar rats after total body 60Co irradiation with lethal doses. Increased activities of cathepsin D in spleen was associated with a marked reduction of splenic weight. The levels of α2M and activities of αM in plasma were both increased with increasing radiation doses, but the increase of α2M levels in spleen was slower and lower than that in liver, kidney and skin. It seems that the effectiveness of α2M in the treatment of rats after total body irradiation might be related with its binding action with cathepsin D and other proteases in spleen and other radiosensitive tissues. One case of acute and two cases of chronic skin radiation injury were treated with α2M preparation, either with or without surgical operation. There were decrease in levels of malonaldehyde as well in activities of cathepsin D, and increase in activities of superoxide dismutase. It suggests that α2M preparation might be useful for both inhibiting excess proteases and scavenging oxygen free radicals

  20. Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Je, Ju Eun; Ahn, Sang Jung; Kim, Na Young; Seo, Jung Soo; Kim, Moo-Sang; Park, Nam Gyu; Kim, Joong Kyun; Chung, Joon Ki; Lee, Hyung Ho

    2009-12-01

    We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3-24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme. PMID:19666132

  1. Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCF10A neoT cells

    International Nuclear Information System (INIS)

    Background. Lysosomal cathepsins B and D have been reported to play a role in various processes leading to progression of malignant disease. In ras-transformed MCF10A neoT cells both enzymes show similar vesicular distribution in perinuclear and peripheral cytoplasmic regions. Results. The co-localization of cathepsins B and D in some vesicles as defined by confocal microscopy supports their co-ordinate activity in the proteolytic cascade. On the other hand, we showed that stefin A, an endogenous intracellular inhibitor of cysteine proteases, did not co-localize with cathepsin B and is presumably not involved in regulation of its enzymatic activity within the vesicles. Intracellular localization of both enzymes was confined to similar vesicles as the fluorescent degradation products of DQ-collagen IV either in individual cells or cell spheroids. The capability of these two enzymes to degrade collagen and other components of extracellular matrix is further supported by the results of Matrigel invasion assay. We showed that specific intracellular (CA-074 Me) and extracellular (CA-074) inhibitors of cathepsin B and pepstatin A, an inhibitor of cathepsin D, significantly reduced invasion of MCF10A neoT cells. Our results also show that in contrast to some other studies the activation peptide of pro-cathepsin D exhibited no mitogenic effect on MCF10A neoT, MCF-7 or HEK-293 cells. Conclusion. We conclude that lysosomal cysteine proteases cathepsins B and D predominantly participate in degradation of extracellular matrix and facilitate invasion of tumour cells. (author)

  2. Structural Basis for Reversible and Irreversible Inhibition of Human Cathepsin L by their Respective dipeptidyl glyoxal and diazomethylketone Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    R Shenoy; J Sivaraman

    2011-12-31

    Cathepsin L plays a key role in many pathophysiological conditions including rheumatoid arthritis, tumor invasion and metastasis, bone resorption and remodeling. Here we report the crystal structures of two analogous dipeptidyl inhibitor complexes which inhibit human cathepsin L in reversible and irreversible modes, respectively. To-date, there are no crystal structure reports of complexes of proteases with their glyoxal inhibitors or complexes of cathepsin L and their diazomethylketone inhibitors. These two inhibitors - inhibitor 1, an {alpha}-keto-{beta}-aldehyde and inhibitor 2, a diazomethylketone, have different groups in the S1 subsite. Inhibitor 1 [Z-Phe-Tyr (OBut)-COCHO], with a Ki of 0.6 nM, is the most potent, reversible, synthetic peptidyl inhibitor of cathepsin L reported to-date. The structure of the inhibitor 1 complex was refined up to 2.2 {angstrom} resolution. The structure of the complex of the inhibitor 2 [Z-Phe-Tyr (t-Bu)-diazomethylketone], an irreversible inhibitor that can inactivate cathepsin L at {micro}M concentrations, was refined up to 1.76 {angstrom} resolution. These two inhibitors have substrate-like interactions with the active site cysteine (Cys25). Inhibitor 1 forms a tetrahedral hemithioacetal adduct, whereas the inhibitor 2 forms a thioester with Cys25. The inhibitor 1 {beta}-aldehyde group is shown to make a hydrogen bond with catalytic His163, whereas the ketone carbonyl oxygen of the inhibitor 2 interacts with the oxyanion hole. tert-Butyl groups of both inhibitors are found to make several non-polar contacts with S' subsite residues of cathepsin L. These studies, combined with other complex structures of cathepsin L, reveal the structural basis for their potency and selectivity.

  3. Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation.

    Science.gov (United States)

    Goulet, Brigitte; Sansregret, Laurent; Leduy, Lam; Bogyo, Matthew; Weber, Ekkehard; Chauhan, Shyam S; Nepveu, Alain

    2007-09-01

    It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus. PMID:17855659

  4. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. PMID:25849458

  5. Impact of the Enhanced Permeability and Retention (EPR Effect and Cathepsins Levels on the Activity of Polymer-Drug Conjugates

    Directory of Open Access Journals (Sweden)

    Amit K. Rajora

    2014-08-01

    Full Text Available Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR effect and (ii the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B. Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates. An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer, although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.

  6. Cathepsin K inhibition reduces CTXII levels and joint pain in the guinea pig model of spontaneous osteoarthritis.

    Science.gov (United States)

    McDougall, J J; Schuelert, N; Bowyer, J

    2010-10-01

    Cathepsin K is a cysteine proteinase which is believed to contribute to osteoarthritis (OA) pathogenesis. This brief report evaluates the effect of the novel selective cathepsin K inhibitor AZ12606133 on cartilage metabolism in the Dunkin-Hartley guinea pig model of spontaneous OA. In parallel, electrophysiological studies were performed to determine whether acute and chronic treatment with the cathepsin K inhibitor could alter joint nociception. Acute treatment of OA knees with AZ12606133 had no effect on joint afferent nerve activity; however, prolonged (1 month) administration of the cathepsin K inhibitor delivered via a chronically implanted osmotic pump significantly reduced mechanosensitivity in response to both non-noxious and noxious joint movements. Urinal concentrations of the cartilage breakdown products cross-linked C-telopeptides of type II collagen (CTXII) were also reduced by chronic cathepsin K inhibition. These data suggest that prolonged AZ12606133 administration can reduce cartilage turnover and joint nociception in the Dunkin-Hartley guinea pig model of spontaneous OA. PMID:20692355

  7. EKSTRAKSI DAN KARAKTERISASI PARSIAL EKSTRAK KASAR ENZIM KATEPSIN DARI IKAN PATIN [Extraction and Partial Characterization of Crude Enzymes Cathepsin from Catfish

    Directory of Open Access Journals (Sweden)

    Muhammad Zakiyul Fikri*

    2014-06-01

    Full Text Available Decomposition of protein by enzymatic process will lead to changes in odor, texture, and appearance of fish. The enzymes that play a role in the enzymatic process is primarily proteolytic enzymes. Cathepsin is one of the proteolytic enzymes found in animal tissue that hydrolyzes peptide bonds of proteins. This study aims to extract the cathepsin, characterize the crude extract derived from catfish. The stages of this research consist of the extraction and characterization of the cathepsin from catfish. Result of the extraction was crude extract of cathepsin with activity of 0.278 U/mL. The enzyme had optimum temperature of 50°C, pH 6 and substrate concentration of 2%. The activity of the cathepsin was inhibited by metal ions of Fe3+, Cu2+, Ca2+, but increased by metal ions of Mg2+.

  8. Inhibition of cathepsin B by E-64 induces oxidative stress and apoptosis in filarial parasite.

    Directory of Open Access Journals (Sweden)

    Mohit Wadhawan

    Full Text Available Current available antifilarial drug strategies only eliminate the larval stages of filarial parasites. Therefore, there is an urgent need of drugs which are macrofilaricidals. Identification of molecular targets crucial for survival of parasite is a prerequisite for drug designing. Cathepsin B, a cysteine protease family member is known to play crucial role in the normal growth, digestion of nutrients, exsheathment of the helminth parasites. Therefore, we targeted this enzyme in the filarial parasite using its specific inhibitor, E-64.We have exposed the parasites to E-64 and observed their motility and viability at various time intervals. It caused marked decrease in the motility and viability of the parasites ultimately leading to their death after 8 hours. It is well known that E-64 protects the cell from apoptosis, however, it causes apoptotic effect in carcinoma cell lines. To understand the mechanism of action of E-64 on parasite survival, we have measured levels of different apoptotic markers in the treated parasites. E-64 significantly reduced the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also activated ced-3, homolog of mammalian caspase 3 suggesting initiation of an apoptotic like event in the filarial parasites. Different antioxidant enzymes were also evaluated to further explore the mechanism behind the death of the parasites. There was marked decrease in the level of GSH and activity of Glutathione reductase and glutathione-s-transferase leading to increased generation of reactive oxygen species. This led to the induced oxidation of fatty acids and protein which might alter the mitochondrial membrane permeability.This study suggests that inhibition of cathepsin B by E-64 generates oxidative stress followed by mitochondrial mediated apoptotic like event in filarial parasites leading to their death. Hence, suggesting filarial cathepsin B as a potential chemotherapeutic target for lymphatic

  9. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  10. Redox-based inactivation of cysteine cathepsins by compounds containing the 4-aminophenol moiety

    OpenAIRE

    Sosič, Izidor; Gobec, Stanislav; Mirković, Bojana; Kos, Janko

    2015-01-01

    Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming. METHODS AND FINDINGS:In this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a k(inact)/K(I) ...

  11. Engineered staphylococcal protein A's IgG-binding domain with cathepsin L inhibitory activity

    International Nuclear Information System (INIS)

    Inhibitory peptide of papain-like cysteine proteases, affinity selected from a random disulfide constrained phage-displayed peptide library, was grafted to staphylococcal protein A's B domain. Scaffold protein was additionally modified in order to allow solvent exposed display of peptide loop. Correct folding of fusion proteins was confirmed by CD-spectroscopy and by the ability to bind the Fc-region of rabbit IgG, a characteristic of parent domain. The recombinant constructs inhibited cathepsin L with inhibitory constants in the low-micromolar range

  12. The p41 isoform of invariant chain is a chaperone for cathepsin L

    OpenAIRE

    Lennon-Duménil, Ana-Maria; Roberts, Rebecca A.; Valentijn, Karine; Driessen, Christoph; Overkleeft, Herman S.; Erickson, Ann; Peters, Peter J.; Bikoff, Elizabeth; Ploegh, Hidde L.; Wolf Bryant, Paula

    2001-01-01

    The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41–CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL a...

  13. IrCL1-The haemoglobinolytic cathepsin L of the hard tick, Ixodes ricinus

    Czech Academy of Sciences Publication Activity Database

    Franta, Zdeněk; Sojka, Daniel; Frantová, Helena; Dvořák, Jan; Horn, Martin; Srba, Jindřich; Talacko, Pavel; Mareš, Michael; Schneider, E.; Craik, C. S.; McKerrow, J. H.; Caffrey, C. R.; Kopáček, Petr

    2011-01-01

    Roč. 41, č. 12 (2011), 1253-1262. ISSN 0020-7519 R&D Projects: GA AV ČR IAA600960910; GA ČR(CZ) GAP207/10/2183; GA ČR GPP502/11/P682; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : tick gut * hemoglobin digestion * cathepsin L Subject RIV: EC - Immunology Impact factor: 3.393, year: 2011

  14. uPAR and cathepsin B inhibition enhanced radiation-induced apoptosis in gliomainitiating cells

    OpenAIRE

    MALLA, RAMA RAO; Gopinath, Sreelatha; Alapati, Kiranmai; Gorantla, Bharathi; Christopher S Gondi; Rao, Jasti S.

    2012-01-01

    Glioblastomas present as diffuse tumors with invasion into normal brain tissue and frequently recur or progress after radiation as focal masses because of glioma-initiating cells. The role of the urokinase-type plasminogen activator receptor (uPAR) and cathepsin B in stem-like phenotype has been extensively studied in several solid tumors. In the present study, we demonstrated that selection of glioma-initiating cells using CD133 expression leads to a specific enrichment of CD133+ cells in bo...

  15. Mutation analysis of cathepsin C gene in a Chinese patient with pre-pubertal periodontitis

    Institute of Scientific and Technical Information of China (English)

    YANG Yuan; BAI Xiao-wen; SONG Shu-juan; GE Li-hong; CAO Cai-fang

    2005-01-01

    @@ Pre-pubertal periodontitis (PPP) is a rare and rapidly progressive form of early onset periodontitis resulting in premature tooth loss of primary and permanent dentitions. Mutations in cathepsin C (CTSC) gene have been found in patients with pre-pubertal periodontitis and Papillon-Lefevre syndrome which also characterized with severe periodontitis and palmoplantar hyperkera-tosis.1-3 To date, more than 40 mutations of CTSC gene have been identified in ethnically diverse people worldwide.4 However, there is no such genetic analysis in China. In the present study, we report the mutation analysis of a Chinese patient with PPP.

  16. CATHEPSIN B EXPRESSION AND ITS RELATIONSHIP WITH MICROVESSEL DENSITY AND BIOLOGICAL BEHAVIOUR OF COLORECTAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    王娅兰; 林晓

    2002-01-01

    Objective: To investigate cathepsin B(CB) expression in colorectal carcinoma and its relationship with microvessel density (MVD) and biological behavior. Methods: CB and MVD were detected by immunohistochemistry in 47 cases of colorectal carcinoma. Results: The expression of CB in mucinous colorectal carcinoma was significantly higher than that in no-mucinous colorectal carcinoma. There was significant difference (P<0.05). The MVD in group with positive CB was stronger than that in group with negative CB. There was also significant difference (P<0.05). Conclusion: The results suggest that CB expression has correlation with MVD, invasion and metastasis in colorectal carcinoma, especially in mucinous colorectal carcinoma.

  17. Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer.

    Science.gov (United States)

    Fan, Wei; Shi, Wen; Zhang, Wenting; Jia, Yinnong; Zhou, Zhengyuan; Brusnahan, Susan K; Garrison, Jered C

    2016-10-01

    This work continues our efforts to improve the diagnostic and radiotherapeutic effectiveness of nanomedicine platforms by developing approaches to reduce the non-target accumulation of these agents. Herein, we developed multi-block HPMA copolymers with backbones that are susceptible to cleavage by cathepsin S, a protease that is abundantly expressed in tissues of the mononuclear phagocyte system (MPS). Specifically, a bis-thiol terminated HPMA telechelic copolymer containing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. Three maleimide modified linkers with different sequences, including cathepsin S degradable oligopeptide, scramble oligopeptide and oligo ethylene glycol, were subsequently synthesized and used for the extension of the HPMA copolymers by thiol-maleimide click chemistry. All multi-block HPMA copolymers could be labeled by (177)Lu with high labeling efficiency and exhibited high serum stability. In vitro cleavage studies demonstrated highly selective and efficient cathepsin S mediated cleavage of the cathepsin S-susceptible multi-block HPMA copolymer. A modified multi-block HPMA copolymer series capable of Förster Resonance Energy Transfer (FRET) was utilized to investigate the rate of cleavage of the multi-block HPMA copolymers in monocyte-derived macrophages. Confocal imaging and flow cytometry studies revealed substantially higher rates of cleavage for the multi-block HPMA copolymers containing the cathepsin S-susceptible linker. The efficacy of the cathepsin S-cleavable multi-block HPMA copolymer was further examined using an in vivo model of pancreatic ductal adenocarcinoma. Based on the biodistribution and SPECT/CT studies, the copolymer extended with the cathepsin S susceptible linker exhibited significantly faster clearance and lower non-target retention without compromising tumor targeting. Overall, these results indicate that

  18. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. PMID:26899824

  19. Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease.

    Science.gov (United States)

    Fox, Christopher; Cocchiaro, Pasquale; Oakley, Fiona; Howarth, Rachel; Callaghan, Krystena; Leslie, Jack; Luli, Saimir; Wood, Katrina M; Genovese, Federica; Sheerin, Neil S; Moles, Anna

    2016-01-01

    During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD. PMID:26831567

  20. Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B activity.

    Science.gov (United States)

    Kim, J-T; Lee, S-J; Kang, M A; Park, J E; Kim, B-Y; Yoon, D-Y; Yang, Y; Lee, C-H; Yeom, Y I; Choe, Y-K; Lee, H G

    2013-01-01

    Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3. PMID:24357805

  1. Separation and identification of cathepsin of non irradiated and irradiated red-fish (Sebates marinus)

    International Nuclear Information System (INIS)

    Cathepsin originated from Sebates marinus muscle hydrolyzed denatured hemoglobin optimally at pH 5. At pH 3 this phenomenon was also detected eventhough with lower intensity. Partial purification by ammonium sulfate fractionation technique was not suitable for the separation of protein contaminants from the active enzyme. By column gel filtration through Sephadex G-75 superfine, fractions with high cathepsin specific activity were recovered i.e. fractions with molecular weights between 15,000-20,000 Daltons. These fractions showed isoelectric components in the pH range of 4.5 - 8 with the predominant protein zones at pH 4.5 - 5. In fish irradiated at a dose of 0.3 Mrad, the gel filtration pattern was not essentially changed. However on focusing the 15,000 - 20,000 Dalton fraction, the isoelectric pattern showed some minor changes, especially the accumulation of protein in the range of pH 4.5 - 5. (author)

  2. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

    2006-01-09

    Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

  3. Cathepsins and their inhibitors as tumor markers in head and neck cancer

    International Nuclear Information System (INIS)

    The invasion and metastasizing of tumor cells is closely connected with the disintegration of basement membranes and extracellular matrix. The carriers of these processes are different proteolytic enzymes, among them cysteine and aspartic cathepsins B, H, L and D as well, a group of ubiquitous lysosomal proteases, and endogenous inhibitors of the former, cystatins. The aim of the present review was to collect the current knowledge on the predictive and prognostic value of cathepsins and their inhibitors in squamous cell carcinoma of the head and neck. In this particular tumor type, the UICC/AJCC TNM-classification system and histopathological characteristics of the tumors were found inadequate to reliably predict either the response to therapy or patients' survival. Moreover, to date, no factor within the wide spectrum of biochemical and histological factors has yet been identified as reliably predicting the natural course of the disease or its response to therapy. To construct a prognostically meaningful tumor profile, new markers are intensively investigated. (author)

  4. The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

    DEFF Research Database (Denmark)

    Mølgaard, Anne; Arnau, Jose; Lauritzen, C.; Larsen, Sine; Petersen, Gitte; Pedersen, John

    2007-01-01

    hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the...

  5. Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease.

    Science.gov (United States)

    Niemer, Melanie; Mehofer, Ulrich; Verdianz, Maria; Porodko, Andreas; Schähs, Philipp; Kracher, Daniel; Lenarcic, Brigita; Novinec, Marko; Mach, Lukas

    2016-03-01

    The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform. PMID:26166069

  6. Bioconjugates of cathepsin K inhibitors with water/soluble polymers: potential drugs for the treatment of osteoporosis and rheumatoid arthritis

    Czech Academy of Sciences Publication Activity Database

    Wang, D.; Pechar, Michal; Kopečková, P.; Brömme, D.; Kopeček, J.

    Istanbul: Hacettepe University Pharmacy Faculty, 2002, s. 12-13. ISBN 975-491-126-6. [International Pharmaceutical Technology Symposium /11./ Intelligent Drug Delivery Systems Better and Safer Therapy. Istanbul (TR), 09.09.2002-11.09.2002] R&D Projects: GA AV ČR KSK4055109 Keywords : Cathepsin K Subject RIV: CE - Biochemistry

  7. Molecular cloning and functional characterisation of a cathepsin L-like proteinases from the fish kinetoplastid parasite Trypanosoma carassii

    NARCIS (Netherlands)

    Ruszczyk, A.; Forlenza, M.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2008-01-01

    Trypanosoma carassii is a fish kinetoplastid parasite that belongs to the family Trypanosomatida. In the present study we cloned a cathepsin L-like proteinase from T. carassii. The nucleotide sequence of 1371 bp translated into a preproprotein of 456 amino acids. The preproprotein contained the oxya

  8. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  9. Structural basis for inhibition of the cathepsin B drug target from the human blood fluke, Schistosoma mansoni

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Řezáčová, Pavlína; Lepšík, Martin; Horn, Martin; Váchová, Jana; Fanfrlík, Jindřich; Brynda, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    Hydra : -, 2012. s. 60-60. [Molecular and Cellular Biology of Helminth Parasites. International Conference /7./. 02.09.2012-07.09.2012, Hydra] R&D Projects: GA MŠk LH12023; GA ČR GA203/09/1585 Institutional support: RVO:61388963 Keywords : cathepsin B1 * Schistosoma * protease * structures Subject RIV: CE - Biochemistry

  10. Single- and Double-Headed Chemical Probes for Detection of Active Cathepsin D in a Cancer Cell Proteome

    Czech Academy of Sciences Publication Activity Database

    Nussbaumerová, Martina; Srp, Jaroslav; Máša, Martin; Hradilek, Martin; Šanda, Miloslav; Reiniš, Milan; Horn, Martin; Mareš, Michael

    2010-01-01

    Roč. 11, č. 11 (2010), s. 1538-1541. ISSN 1439-4227 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : cathepsin D * cancer * activity-based probes Subject RIV: CE - Biochemistry Impact factor: 3.945, year: 2010

  11. Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting.

    Science.gov (United States)

    Flanagan-Steet, Heather; Aarnio, Megan; Kwan, Brian; Guihard, Pierre; Petrey, Aaron; Haskins, Mark; Blanchard, Frederic; Steet, Richard

    2016-03-01

    Hypersecretion of acid hydrolases is a hallmark feature of mucolipidosis II (MLII), a lysosomal storage disease caused by loss of carbohydrate-dependent lysosomal targeting. Inappropriate extracellular action of these hydrolases is proposed to contribute to skeletal pathogenesis, but the mechanisms that connect hydrolase activity to the onset of disease phenotypes remain poorly understood. Here we link extracellular cathepsin K activity to abnormal bone and cartilage development in MLII animals by demonstrating that it disrupts the balance of TGFß-related signaling during chondrogenesis. TGFß-like Smad2,3 signals are elevated and BMP-like Smad1,5,8 signals reduced in both feline and zebrafish MLII chondrocytes and osteoblasts, maintaining these cells in an immature state. Reducing either cathepsin K activity or expression of the transcriptional regulator Sox9a in MLII zebrafish significantly improved phenotypes. We further identify components of the large latent TGFß complex as novel targets of cathepsin K at neutral pH, providing a possible mechanism for enhanced Smad2,3 activation in vivo. These findings highlight the complexity of the skeletal disease associated with MLII and bring new insight to the role of secreted cathepsin proteases in cartilage development and growth factor regulation. © 2015 American Society for Bone and Mineral Research. PMID:26404503

  12. Cathepsin L Plays a Major Role in Cholecystokinin Production in Mouse Brain Cortex and in Pituitary AtT-20 Cells: Protease Gene Knockout and Inhibitor Studies

    Science.gov (United States)

    Beinfeld, Margery C.; Funkelstein, Lydiane; Foulon, Thierry; Cadel, Sandrine; Kitagawa, Kouki; Toneff, Thomas; Reinheckel, Thomas; Peters, Christoph; Hook, Vivian

    2009-01-01

    Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK0/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells. PMID:19589362

  13. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.

    Science.gov (United States)

    Zhang, T; Rawson, D M; Tosti, L; Carnevali, O

    2008-04-01

    This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk

  14. RNAi-Mediated Simultaneous Downregulation of uPAR and Cathepsin B Induces Caspase 8-Mediated Apoptosis in SNB19 Human Glioma Cells

    OpenAIRE

    Christopher S Gondi; Kandhukuri, Neelima; Kondraganti, Shakuntala; Gujrati, Meena; Olivero, William C.; Dinh, Dzung H.; Rao, Jasti S.

    2006-01-01

    The invasive character of gliomas depends on proteolytic cleavage of the surrounding extracellular matrix. Cathepsin B and uPAR together are known to be overexpressed in gliomas, and as such, are attractive targets for gene therapy. In the present study, we used plasmid constructs to induce the RNAi-mediated downregulation of uPAR and Cathepsin B in SNB19 human glioma cells. We observed that the simultaneous downregulation of uPAR and Cathepsin B induces the up-regulation of pro-apoptotic gen...

  15. Expressional induction of Paralichthys olivaceus cathepsin B gene in response to virus, poly I:C and lipopolysaccharide.

    Science.gov (United States)

    Zhang, Fu-Tie; Zhang, Yi-Bing; Chen, Yu-Dong; Zhu, Rong; Dong, Cai-Wen; Li, Yang-Yang; Zhang, Qi-Ya; Gui, Jian-Fang

    2008-11-01

    Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In this study, Paralichthys olivaceus cathepsin B (PoCatB) cDNA was isolated from flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and subsequently identified as a virally induced gene. The full length cDNA of PoCatB is 1801bp encoding 330-amino acids. The deduced protein has high homology to all known cathepsin B proteins, containing an N-terminal signal peptide, cysteine protease active sites, the occluding loop segment and a glycosylation site, all of which are conserved in the cathepsin B family. PoCatB transcription of FEC cells could be induced by turbot (Scophthalmus maximus) rhabdovirus (SMRV), UV-inactivated SMRV, UV-inactivated GCHV, poly I:C or lipopolysaccharide (LPS), and SMRV or poly I:C was revealed to be most effective among the five inducers. In normal flounder, PoCatB mRNA was detectable in all examined tissues. Moreover, SMRV infection could result in significant upregulation of PoCatB mRNA, predominantly in spleen, head kidney, posterior kidney, intestine, gill and muscle with 18.2, 10.9, 24.7, 12, 31.5 and 18 fold increases at 72h post-infection respectively. These results provided the first evidence for the transcriptional induction of cathepsin B in fish by virus and LPS, indicating existence of a novel function in viral defense. PMID:18755278

  16. Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach

    Science.gov (United States)

    Ferraro, Florencia; Merlino, Alicia; dell´Oca, Nicolás; Gil, Jorge; Tort, José F.; Gonzalez, Mercedes; Cerecetto, Hugo; Cabrera, Mauricio

    2016-01-01

    Background Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections. Methodology/Principle Findings We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1). Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 μM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells. Conclusions/Significance Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34) is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues

  17. Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

    Directory of Open Access Journals (Sweden)

    Florencia Ferraro

    2016-07-01

    Full Text Available Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections.We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1. Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 μM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells.Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34 is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control

  18. Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes

    Science.gov (United States)

    Löser, Reik; Pietzsch, Jens

    2015-06-01

    Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge towards their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

  19. Targeting Cathepsin E in Pancreatic Cancer by a Small Molecule Allows In Vivo Detection

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    Edmund J. Keliher

    2013-07-01

    Full Text Available When resectable, invasive pancreatic ductal adenocarcinoma (PDAC is most commonly treated with surgery and radiochemotherapy. Given the intricate local anatomy and locoregional mode of dissemination, achieving clean surgical margins can be a significant challenge. On the basis of observations that cathepsin E (CTSE is overexpressed in PDAC and that an United States Food and Drug Administration (FDA-approved protease inhibitor has high affinity for CTSE, we have developed a CTSE optical imaging agent [ritonavir tetramethyl-BODIPY (RIT-TMB] for potential intraoperative use.We show nanomolar affinity [half maximal inhibitory concentration (IC50 of 39.9 ± 1.2 nM] against CTSE of the RIT-TMB in biochemical assays and intracellular accumulation and target-to-background ratios that allow specific delineation of individual cancer cells. This approach should be useful for more refined surgical staging, planning, and resection with curative intent.

  20. Running-Induced Systemic Cathepsin B Secretion Is Associated with Memory Function.

    Science.gov (United States)

    Moon, Hyo Youl; Becke, Andreas; Berron, David; Becker, Benjamin; Sah, Nirnath; Benoni, Galit; Janke, Emma; Lubejko, Susan T; Greig, Nigel H; Mattison, Julie A; Duzel, Emrah; van Praag, Henriette

    2016-08-01

    Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. In vivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition. PMID:27345423

  1. Expression of cathepsin B is related to tumorigenicity of breast cancer cell lines

    International Nuclear Information System (INIS)

    Background. The lysosomal cysteine proteases cathepsins B (CatB) and L (CatL) and their endogenous inhibitors, stefins A (StA) and B (StB), are widely thought to be involved in the progression of human breast carcinoma. Previously we showed that, in model breast carcinoma cell lines, the reported tumorigenicity was not directly related to their in vitro invasive potential. However, CatL expression was positively related to the invasiveness of the cells and inversely related to the levels of StA. Here we challenge the hypothesis that imbalance between CatB and the two stefins is associated either with the invasiveness or the reported tumorigenicity of the panel of selected breast carcinoma cells. Results. We investigated levels of mRNA, protein and activity for CatB in the panel of human breast carcinoma cell lines whose tumorigenicity in vivo increased in the order MCF-7 < MDA-MB468 < MDAMB231 < MDA-MB435, the most invasive being MDA-231. Levels of expression of mRNA, protein and activity for CatB were highly correlated and increased progressively with cell tumorigenicity. The ratio of CatB to stefins was shifted in favour of CatB in the more tumorigenic cell lines. Conclusions. Since CatL has been shown previously to be associated with invasive potential and, in this study, CatB expression was found positively associated with the tumorigenicity of the same breast carcinoma cell lines, the two cathepsins in these cells do not appear to be regulated in a coordinated manner. CatB expression and the ratio between CatB and stefins increased progressively with tumorigenicity of the cells and suggests a similar situation in human tumours in vivo. (author)

  2. Overexpression of cathepsin K during silica-induced lung fibrosis and control by TGF-β

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    Lison Dominique

    2005-07-01

    Full Text Available Abstract Background Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s (Cat are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis. Methods Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts. Results In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-β expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-β1. Conclusion Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-β appears to limit its overexpression in response to silica particles.

  3. Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells.

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    Ira O Racoma

    Full Text Available Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II. TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a

  4. Expression and prognostic significance of Cathepsin L in early cutaneous malignant melanoma.

    Science.gov (United States)

    Stabuc, B; Mrevlje, Z; Markovic, J; Stabuc-Silih, M

    2006-01-01

    Cathepsins are papain-like lysosome cysteine proteases involved in tumor growth, invasiveness and spread, angiogenesis and alteration in immune and inflammatory responses. We investigated the differences in cathepsin L (CatL) concentrations in primary cutaneous malignant melanoma stage I and normal skin and correlated these values with well-established malignant melanoma prognostic factors. The study was performed on 36 patients (17 men and 19 women; mean age 54 years; range 21-84 years) with histological confirmed primary malignant melanomas less than 1.5 mm thick. The CatL concentrations were measured in 36 pairs of triton extracts of cytosols prepared from the tumor and adjacent normal tissue samples (matched pairs). The CatL concentrations were determined by commercially available enzyme-liked immunosorbent (ELISA) assay from KRKA (Novo Mesto, Slovenia). Significantly higher concentrations of CatL were detected in malignant melanomas than in normal surrounding skin (6.73 vs. 1.42 ng/mg total protein (mgp), pskin concentrations for CatL were found. The malignant melanoma CatL concentrations correlated significantly with normal skin (r=0.38; p=0.021). CatL concentrations were significantly lower (p0.75 mm, Clark invasion of >or=II and

  5. THE PROGNOSIS SIGNIFICANCE OF CATHEPSIN-D EXPRESSION IN THE DIFFERENT LOCATIONS IN AXILLARY NODES NEGATIVE CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    NIU; Yun

    2001-01-01

    [1]Garcia M, Platet N, Liaudet Estradiol, et al. Biological and clinical significance of cathepsin D in breast cancer metastasis [J]. Stem Cells 1996; 14:642.[2]Johnson MD, Torri JA, Lippman ME, et al. The role of cathepsin D in the invasiveness of human breast cancer cells [J]. Cancer Res 1993; 53: 873.[3]Duffy MJ. Proteases as prognostic markers in cancer [J]. Clin Cancer Res 1996; 2:613.[4]Westley BR, May FE. Cathepsin D and breast cancer [J]. Eur J Cancer 1996; 32A:7.[5]Riley LB, Lange MK, Browne RJ, et al. Analysis of cathepsin D in human breast cancer: usefulness of the processed 31 kDa active form of the enzyme as a prognostic indicator in node-negative and node-positive patients [J]. Breast Cancer Res Treat 2000; 60:173.[6]Fu XL. Histopathologic diagnosis. Chinese Common Malignant Tumor Diagnosis and Treatment Rule. Breast Carcinoma Volume [M]. 2nd ed. Beijing: Beijing Medical University and Chinese Xiehe Medical University Union Publisher, 1999; 23.[7]Yang SQ. Health Statistics [M]. 3rd ed. Beijing: People Health Publisher, 1998; 131.[8]Bittl A, Nap M, Jager W, et al. Immuno-histochemical detection of P-glycoprotein on frozen and paraffin-embedded tissue sections of normal and malignant tissues [J]. Anticancer Res 1995; 15:1007.[9]Isola J, Weitz S, Visakorpi T, et al. Cathepsin D expression detected by immunohistochemistry has independent prognostic value in axillary node-negative breast cancer [J]. J Clin Oncol 1993; 11:36.[10]Castiglioni T, Merino MJ, Elsner B, et al. Immunohistochemical analysis of cathepsins D, B, and L in human breast cancer [J]. Hum Pathol 1994; 25:857.[11]Montcourrier P, Mangeat PH, Valembois C, et al. Characterization of very acidic phagosomes in breast cancer cells and their association with invasion [J]. J Cell Sci 1994; 107:238l.[12]Foekens JA, Look MP, Bolt de Vries J, et al. Cathepsin-D in primary breast cancer: prognostic evaluation involving 2810 patients [J]. Br J Cancer 1999

  6. Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions

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    Brömme Dieter

    2009-09-01

    Full Text Available Abstract Background Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. Results The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. Conclusion The results revealed distinct cleavage patterns

  7. THE ROLE OF CYTOKINE SYSTEM RANKL-RANK-OPG AND CATHEPSIN K IN THE PATHOGENESIS OF OSTEOPOROSIS: ACHIEVEMENTS AND PERSPECTIVES IN THE TREATMENT OF DISEASE

    OpenAIRE

    S. Sagalovsky; Kunze, P.; M. Schönert

    2012-01-01

    The article presents review of literature dedicated to the contemporary view on the cellular-molecular mechanisms of the bone remodeling and pathogenesis of the osteoporosis. The discovery of the cytokine RANKL-RANK-OPG system and significant role of the cathepsin K in process bone remodeling has made progress in understanding the mechanisms development disease and possible to development drugs of the new generation – denosumab, a fully human RANKL monoclonal antibody and inhibitor cathepsin ...

  8. Expression of Cathepsin L in tumor cells and tumor-associated macrophages in patients with Ewing sarcoma family of tumors: A pilot study

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    Bivas Biswas

    2015-01-01

    Full Text Available Background: Cysteine protease Cathepsin L is involved in bone remodeling and expressed in activated macrophages. It is highly expressed in metastatic tumor tissue, especially with bone metastases. Aims: We evaluated immunohistochemical expression of Cathepsin L in tumor cells and tumor-associated macrophages (TAMs in chemo-naive Ewing sarcoma. Settings and Design: Retrospective evaluation of archived specimens of Ewing sarcoma. Materials and Methods: Immunohistochemical staining was performed on archived blocks of chemo-naive patients with Ewing sarcoma treated with uniform chemotherapy at our institute between January 2009 and November 2011. Statistical Analysis: Immunohistochemical expression was co-related with baseline demographics and survival. Results: During the study period, we had evaluable baseline samples from 62 patients with median age 15 years (range: 2-40; 26 (42% had metastases. Cathepsin L expression in tumor cells was observed in 8/62 (13% specimens. None of the baseline clinical characteristics correlated with Cathepsin L expression. Cathepsin L positivity was associated with poor response to neoadjuvant chemotherapy (NACT (P = 0.05, but did not influence either event-free-survival (EFS or overall survival. Cathepsin L was expressed in TAMs in all specimens. Grade 3 TAMs (>10 TAMs/high power field was associated with better response to NACT (P = 0.05. On univariate analysis Grade 3 TAMs predicted superior EFS (median EFS 28.5 months in those with Grade 3 TAMs versus 14.8 months in those with grade ½ TAMs [P = 0.04]. Conclusions: Cathepsin L expression by immunohistochemistry was low in our patient cohort, and it did not affect the outcome. In addition, Grade 3 TAMs with Cathepsin L expression was associated with improved EFS.

  9. A candidate gene approach to identify DNA markers associated with meat quality and production traits: investigation of several cathepsin genes in Italian heavy pigs.

    OpenAIRE

    Speroni, Camilla

    2010-01-01

    The cathepsin enzymes represent an important family of lysosomal proteinases with a broad spectrum of functions in many, if not in all, tissues and cell types. In addition to their primary role during the normal protein turnover, they possess highly specific proteolytic activities, including antigen processing in the immune response and a direct role in the development of obesity and tumours. In pigs, the involvement of cathepsin enzymes in proteolytic processes have important effects duri...

  10. Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

    Science.gov (United States)

    Lautwein, Alfred; Kraus, Marianne; Reich, Michael; Burster, Timo; Brandenburg, J; Overkleeft, Herman S; Schwarz, Gerold; Kammer, Winfried; Weber, Ekkehard; Kalbacher, Hubert; Nordheim, Alfred; Driessen, Christoph

    2004-05-01

    Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner. PMID:14966190

  11. A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci.

    Directory of Open Access Journals (Sweden)

    Martin A Bewley

    Full Text Available The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

  12. Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression.

    Science.gov (United States)

    Gnirss, Kerstin; Kühl, Annika; Karsten, Christina; Glowacka, Ilona; Bertram, Stephanie; Kaup, Franziska; Hofmann, Heike; Pöhlmann, Stefan

    2012-03-01

    Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation. PMID:22222211

  13. Expression of schistosomal cathepsin l1 in Escherichia coli and evaluation of its protective capacity in an animal challenge

    Directory of Open Access Journals (Sweden)

    PA Miyasato

    2009-01-01

    Full Text Available Schistosomes utilize proteinases to accomplish several activities such as tissue penetration, tissue digestion and evasion of host immune responses. Cathepsin L is a cysteine proteinase of the papain superfamily detected in their gut lumen which indicates that this enzyme contributes to the proteolysis of ingested hemoglobin. Due to the roles played in the schistosome biology, proteolytic enzymes are considered potential targets for developing and guiding antischistosomal therapies. In the present work, the cathepsin L1 cDNA coding of Schistosoma mansoni was cloned into the pAE vector that provides high-level expression of heterologous proteins in Escherichia coli. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions through nickel-charged chromatography and used for experimental animal vaccination. ELISA was performed with the pooled sera. Although this protein was shown to be immunogenic, mice immunized with three doses of recombinant protein plus aluminum hydroxide as adjuvant were not protected against S. mansoni infection.

  14. Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner.

    OpenAIRE

    Orhan Rasid; Véronique Mériaux; Erin M Khan; Chloé Borde; Ioana S Ciulean; Catherine Fitting; Bénédicte Manoury; Jean-Marc Cavaillon; Noëlle Doyen

    2016-01-01

    A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. U...

  15. CCL2 is transcriptionally controlled by the lysosomal protease cathepsin S in a CD74-dependent manner

    OpenAIRE

    Wilkinson, Richard; Magorrian, Sinead; Williams, Richard; Young, Andrew; Small, Donna; Scott, Christopher; Burden, Roberta

    2015-01-01

    Cathepsins S (CatS) has been implicated in numerous tumourigenic processes and here we document for the first time its involvement in CCL2 regulation within the tumour microenvironment. Analysis of syngeneic tumours highlighted reduced infiltrating macrophages in CatS depleted tumours. Interrogation of tumours and serum revealed genetic ablation of CatS leads to the depletion of several pro-inflammatory chemokines, most notably, CCL2. This observation was validated in vitro, where shRNA deple...

  16. Frequency of CFTR, SPINK1, and Cathepsin B Gene Mutation in North Indian Population: Connections between Genetics and Clinical Data

    OpenAIRE

    Shweta Singh; Gourdas Choudhuri; Sarita Agarwal

    2014-01-01

    Objectives. Genetic mutations and polymorphisms have been correlated with chronic pancreatitis (CP). This study aims to investigate the association of genetic variants of cystic fibrosis transmembrane conductance regulator (CFTR) and serine protease inhibitor Kazal type 1 (SPINK-1) genes and Cathepsin B gene polymorphisms with CP and to associate genetic backgrounds with clinical phenotypes. Methods. 150 CP patients and 150 normal controls were enrolled consecutively. We analyzed SPINK-1 N34S...

  17. Functional expression and characterization of cathepsin B and L from the gut of the tick Ixodes ricinus

    Czech Academy of Sciences Publication Activity Database

    Franta, Zdeněk; Pěničková, Helena; Dvorak, J.; Schneider, E. I.; Horn, Martin; Mareš, Michael; Sojka, Daniel; McKerrow, J. H.; Caffrey, C. R.; Kopáček, Petr

    2009-01-01

    Roč. 276, S1 (2009), s. 309-309. ISSN 1742-464X. [34th FEBS Congress: Life's Molecular Interactions. 04.07.2009-09.07.2009, Prague] R&D Projects: GA AV ČR IAA600960910; GA MŠk(CZ) LC06009 Institutional research plan: CEZ:AV0Z60220518; CEZ:AV0Z40550506 Keywords : cathepsin B and L * Ixodes ricinus * functionl expresssion Subject RIV: EB - Genetics ; Molecular Biology

  18. IGFBP-4 Anti-Angiogenic and Anti-Tumorigenic Effects Are Associated with Anti-Cathepsin B Activity

    OpenAIRE

    María J. Moreno; Marguerite Ball; Marina Rukhlova; Jacqueline Slinn; Denis L'Abbe; Umar Iqbal; Robert Monette; Martin Hagedorn; Maureen D O'Connor-McCourt; Yves Durocher; Stanimirovic, Danica B

    2013-01-01

    Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our stu...

  19. IGFBP-4 Anti-Angiogenic and Anti-Tumorigenic Effects Are Associated with Anti-Cathepsin B Activity1

    OpenAIRE

    María J. Moreno; Ball, Marguerite; Rukhlova, Marina; Slinn, Jacqueline; L'Abbe, Denis; Iqbal, Umar; Monette, Robert; Hagedorn, Martin; O'Connor-McCourt, Maureen D.; Durocher, Yves; Stanimirovic, Danica B

    2013-01-01

    Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our stu...

  20. Progress in Cathepsin K Inhibitor Odanacatib%组织蛋白酶K抑制剂Odanacatib研究进展

    Institute of Scientific and Technical Information of China (English)

    晁爱军

    2014-01-01

    骨质疏松症患病率近年来明显增加。破骨细胞功能活化是骨质疏松发生的重要原因。组织蛋白酶是破骨细胞表达的一类酶,参与有机质的降解,与骨质疏松的发生密切相关。其中组织蛋白酶K是最重要的一种酶,组织蛋白酶K抑制剂Odanacatib(ODN)可以通过抑制其对有机质的降解治疗骨质疏松。在目前进行的一些研究中发现,应用ODN可以增加皮质厚度和小梁骨的骨矿含量,进而增加骨密度和骨的负荷强度。本文就ODN的药理特性、动物实验及临床研究的进展进行综述。%The prevalence of osteoporosis has increased during recent years. The activation in the function of osteo⁃clast is the main reason of osteoporosis. Cathepsin is expressed by osteoclast and involved in the degradation of collagen of bone,and causes osteoporosis. Cathepsin K is a kind of most important enzyme in the family of cathepsin. Odanacatib(ODN) is the inhibitor of cathepsin K, and it may be used to anti-osteoporosis thought inhibiting the degradation of bone collagen. It was found that the cortical thickness and bone minerals of cancellous increased after taking ODN in the studies, and then the density and the strength of bone increased. This study reviewed the pharmacological profile of ODN and the progresses of ani⁃mal study and clinical trials about ODN.

  1. BRCA1 loss activates cathepsin L–mediated degradation of 53BP1 in breast cancer cells

    OpenAIRE

    Grotsky, David A.; González-Suárez, Ignacio; Novell Álvarez, Anna; Neumann, Martin; Yaddanapudi, Sree C.; Croke, Monica; Martínez Alonso, Montserrat; Redwood, Abena B.; Ortega-Martinez, Sylvia; Feng, Zhihui; Lerma, Enrique; Ramon y Cajal, Teresa; Zhang, Junran; Matias-Guiu, Xavier; Dusso, Adriana

    2013-01-01

    Loss of 53BP1 rescues BRCA1 deficiency and is associated with BRCA1-deficient and triple-negative breast cancers (TNBC) and with resistance to genotoxic drugs. The mechanisms responsible for decreased 53BP1 transcript and protein levels in tumors remain unknown. Here, we demonstrate that BRCA1 loss activates cathepsin L (CTSL)–mediated degradation of 53BP1. Activation of this pathway rescued homologous recombination repair and allowed BRCA1-deficient cells to bypass growth arrest. Importantly...

  2. Sickle Cell Disease Activates Peripheral Blood Mononuclear Cells to Induce Cathepsins K and V Activity in Endothelial Cells

    OpenAIRE

    Platt, Manu O.; Sindhuja Surapaneni; Keegan, Philip M.

    2012-01-01

    Sickle cell disease is a genetic disease that increases systemic inflammation as well as the risk of pediatric strokes, but links between sickle-induced inflammation and arterial remodeling are not clear. Cathepsins are powerful elastases and collagenases secreted by endothelial cells and monocyte-derived macrophages in atherosclerosis, but their involvement in sickle cell disease has not been studied. Here, we investigated how tumor necrosis alpha (TNFα) and circulating mononuclear cell adhe...

  3. Cathepsin D protects colorectal cancer cells from acetate-induced apoptosis through autophagy-independent degradation of damaged mitochondria

    OpenAIRE

    Oliveira, Cláudia S. F.; Pereira, Helena Paula Fernandes; Alves, Sara Cristina Sequeira; Castro, Lisandra Marisa Flores; Baltazar, Fátima; Chaves, Susana; Preto, Ana; Côrte-Real, Manuela

    2015-01-01

    Acetate is a short-chain fatty acid secreted by Propionibacteria from the human intestine, known to induce mitochondrial apoptotic death in colorectal cancer (CRC) cells. We previously established that acetate also induces lysosome membrane permeabilization in CRC cells, associated with release of the lysosomal protease cathepsin D (CatD), which has a well-established role in the mitochondrial apoptotic cascade. Unexpectedly, we showed that CatD has an antiapoptotic role in this process, as p...

  4. Functional analysis of the cathepsin-like cysteine protease genes in adult Brugia malayi using RNA interference.

    Directory of Open Access Journals (Sweden)

    Louise Ford

    Full Text Available BACKGROUND: Cathepsin-like enzymes have been identified as potential targets for drug or vaccine development in many parasites, as their functions appear to be essential in a variety of important biological processes within the host, such as molting, cuticle remodeling, embryogenesis, feeding and immune evasion. Functional analysis of Caenorhabditis elegans cathepsin L (Ce-cpl-1 and cathepsin Z (Ce-cpz-1 has established that both genes are required for early embryogenesis, with Ce-cpl-1 having a role in regulating in part the processing of yolk proteins. Ce-cpz-1 also has an important role during molting. METHODS AND FINDINGS: RNA interference assays have allowed us to verify whether the functions of the orthologous filarial genes in Brugia malayi adult female worms are similar. Treatment of B. malayi adult female worms with Bm-cpl-1, Bm-cpl-5, which belong to group Ia of the filarial cpl gene family, or Bm-cpz-1 dsRNA resulted in decreased numbers of secreted microfilariae in vitro. In addition, analysis of the intrauterine progeny of the Bm-cpl-5 or Bm-cpl Pro dsRNA- and siRNA-treated worms revealed a clear disruption in the process of embryogenesis resulting in structural abnormalities in embryos and a varied differential development of embryonic stages. CONCLUSIONS: Our studies suggest that these filarial cathepsin-like cysteine proteases are likely to be functional orthologs of the C. elegans genes. This functional conservation may thus allow for a more thorough investigation of their distinct functions and their development as potential drug targets.

  5. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    International Nuclear Information System (INIS)

    Research highlights: → Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. → CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. → CB-deficiency significantly increased the mean survival ratio of injured neurons. → Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy-induced mortor neuron

  6. 6-Shogaol has anti-amyloidogenic activity and ameliorates Alzheimer's disease via CysLT1R-mediated inhibition of cathepsin B.

    Science.gov (United States)

    Na, Ji-Young; Song, Kibbeum; Lee, Ju-Woon; Kim, Sokho; Kwon, Jungkee

    2016-08-12

    Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, the effects of 6-shogaol on Alzheimer's disease (AD) have not yet been investigated. Here we aimed to determine whether 6-shogaol exerts neuroprotective effects against AD. Specifically, we investigated the effects of 6-shogaol on the cysteinyl leukotriene 1 receptor (CysLT1R), a major factor in AD pathogenesis. Moreover, we clarified the relationship between CysLT1R and cathepsin B, a cysteine protease. We used in vitro and in vivo models to determine whether 6-shogaol inhibits CysLT1R/cathepsin B in an amyloid-beta (Aβ; 1-42)-induced model of neurotoxicity. We first confirmed that CysLT1R and cathepsin B are upregulated by Aβ (1-42) and that CysLT1R activation induces cathepsin B. In contrast, we found that 6-shogaol-mediated inhibition of CysLT1R downregulates cathepsin B in both in vitro and in vivo models. Furthermore, we found that 6-shogaol-mediated inhibition of CysLT1R/cathepsin B reduces Aβ deposition in the brain and ameliorates behavioral deficits in APPSw/PS1-dE9 Tg mice. Our results indicate that 6-shogaol is a CysLT1R/cathepsin B inhibitor and is a novel potential therapeutic agent for the treatment of various neurodegenerative diseases, including AD. PMID:27286707

  7. Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.

    Directory of Open Access Journals (Sweden)

    Tien-Sheng Tseng

    Full Text Available Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB. Far-UV circular dichroism (CD, intrinsic fluorescence, and nuclear magnetic resonance (NMR spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.

  8. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

    Science.gov (United States)

    Garcia-Campos, Andres; Cwiklinski, Krystyna; Dalton, John P.; Hokke, Cornelis H.; O’Neill, Sandra; Mulcahy, Grace

    2016-01-01

    Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F

  9. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans.

    Science.gov (United States)

    Garcia-Campos, Andres; Ravidà, Alessandra; Nguyen, D Linh; Cwiklinski, Krystyna; Dalton, John P; Hokke, Cornelis H; O'Neill, Sandra; Mulcahy, Grace

    2016-05-01

    Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F

  10. Detection of oral squamous cell carcinoma metastasis with cathepsin D: An immunohistochemical approach

    Directory of Open Access Journals (Sweden)

    Seema Kapoor

    2014-01-01

    Full Text Available Background: The lysosomal protease cathepsin D (CD has been associated with tumor progression in malignant tumors including oral squamous cell carcinoma (OSCC. The purpose of this study was to find out any association between the CD and lymph node metastasis and to study the correlation of CD with various clinicopathological parameters to aid in assessment of its role as a prognostic indicator. Materials and Methods: Immunohistochemical staining was performed on 20 OSCC samples with polyclonal antibody against CD. Positive results indicative of the presence of CD were further analyzed to determine any correlation between the CD and other clinicopathological parameters. Pearson Chi-square analyses, Spearsman correlation coefficient, Mann-Whitney test, Kruskal Wallis test and student t test were used for statistical analysis (P < 0.05. Results: Patients with lymph node metastasis showed statistically significant increase in CD expression (P < 0.01. Increasing tumor size seemed to correlate with increased CD expression (P < 0.05. Conclusion: Based on its association with other clinicopathological variables, CD expression can be used for the assessment of patient survival in cases of OSCC.

  11. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  12. Cathepsin S expression: An independent prognostic factor in glioblastoma tumours--A pilot study.

    Science.gov (United States)

    Flannery, Thomas; McQuaid, Stephen; McGoohan, Caroline; McConnell, Robert S; McGregor, Gordon; Mirakhur, Meenakshi; Hamilton, Peter; Diamond, James; Cran, Gordon; Walker, Brian; Scott, Christopher; Martin, Lorraine; Ellison, David; Patel, Chirag; Nicholson, Clare; Mendelow, David; McCormick, Derek; Johnston, Patrick G

    2006-08-15

    Cysteine proteinases have been implicated in astrocytoma invasion. We recently demonstrated that cathepsin S (CatS) expression is up-regulated in astrocytomas and provided evidence for a potential role in astrocytoma invasion (Flannery et al., Am J Path 2003;163(1):175-82). We aimed to evaluate the significance of CatS in human astrocytoma progression and as a prognostic marker. Frozen tissue homogenates from 71 patients with astrocytomas and 3 normal brain specimens were subjected to ELISA analyses. Immunohistochemical analysis of CatS expression was performed on 126 paraffin-embedded tumour samples. Fifty-one astrocytoma cases were suitable for both frozen tissue and paraffin tissue analysis. ELISA revealed minimal expression of CatS in normal brain homogenates. CatS expression was increased in grade IV tumours whereas astrocytoma grades I-III exhibited lower values. Immunohistochemical analysis revealed a similar pattern of expression. Moreover, high-CatS immunohistochemical scores in glioblastomas were associated with significantly shorter survival (10 vs. 5 months, p = 0.014). With forced inclusion of patient age, radiation dose and Karnofsky score in the Cox multivariate model, CatS score was found to be an independent predictor of survival. CatS expression in astrocytomas is associated with tumour progression and poor outcome in glioblastomas. CatS may serve as a useful prognostic indicator and potential target for anti-invasive therapy. PMID:16550604

  13. Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

    Science.gov (United States)

    Tjondrokoesoemo, Andoria; Schips, Tobias G; Sargent, Michelle A; Vanhoutte, Davy; Kanisicak, Onur; Prasad, Vikram; Lin, Suh-Chin J; Maillet, Marjorie; Molkentin, Jeffery D

    2016-05-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and β-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD. PMID:26966179

  14. Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

    International Nuclear Information System (INIS)

    Highlights: ► Cat S is highly expressed in HCC cells with high metastatic potential. ► Knockdown of Cat S inhibits growth and invasion of HCC cells. ► Knockdown of Cat S inhibits HCC-associated angiogenesis. ► Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

  15. Silencing cathepsin S gene expression inhibits growth, invasion and angiogenesis of human hepatocellular carcinoma in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Qi; Wang, Xuedi; Zhang, Hanguang; Li, Chuanwei [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China); Fan, Junhua [Department of Gastroenterology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China); Xu, Jing, E-mail: jxuapr@yahoo.com.cn [Department of Hepatobiliary and Vascular Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021 (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Cat S is highly expressed in HCC cells with high metastatic potential. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits growth and invasion of HCC cells. Black-Right-Pointing-Pointer Knockdown of Cat S inhibits HCC-associated angiogenesis. Black-Right-Pointing-Pointer Cat S might be a potential target for HCC therapy. -- Abstract: Cathepsin S (Cat S) plays an important role in tumor invasion and metastasis by its ability to degrade extracellular matrix (ECM). Our previous study suggested there could be a potential association between Cat S and hepatocellular carcinoma (HCC) metastasis. The present study was designed to determine the role of Cat S in HCC cell growth, invasion and angiogenesis, using RNA interference technology. Small interfering RNA (siRNA) sequences for the Cat S gene were synthesized and transfected into human HCC cell line MHCC97-H. The Cat S gene targeted siRNA-mediated knockdown of Cat S expression, leading to potent suppression of MHCC97-H cell proliferation, invasion and angiogenesis. These data suggest that Cat S might be a potential target for HCC therapy.

  16. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    Science.gov (United States)

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods. PMID:15797237

  17. Excision of foreign gene product with cathepsin D in chicken hepatoma cell line

    International Nuclear Information System (INIS)

    To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24 h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens

  18. Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer.

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    Cui, F; Wang, W; Wu, D; He, X; Wu, J; Wang, M

    2016-07-01

    Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC. PMID:26474873

  19. Optical Imaging of Cancer-Related Proteases Using Near-Infrared Fluorescence Matrix Metalloproteinase-Sensitive and Cathepsin B-Sensitive Probes

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    Ji Young Yhee, Sun Ah Kim, Heebeom Koo, Sohee Son, Ju Hee Ryu, In-Chan Youn, Kuiwon Choi, Ick Chan Kwon, Kwangmeyung Kim

    2012-01-01

    Full Text Available Cathepsin B and matrix metalloproteinase (MMP play key roles in tumor progression by controlled degradation of extracellular matrix. Consequently, these proteases have been attracted in cancer research, and many imaging probes utilizing these proteases have been developed. Our groups developed cathepsin B and MMP imaging nanoprobes based on polymer nanoparticle platform. Both cathepsin B and MMP imaging probes used near-infrared fluorescence (NIRF dye and dark-quencher to for high sensitivity, and protease-sensitive peptide sequence in each probe authorized high specificity of the probes. We compared the bioactivities of cathepsin B and MMP sensitive probes in cancer-related environments to investigate the biological property of the probes. As a result, cathepsin B probe showed fluorescence recovery after the probe entered the cytoplasm. This property could be useful to evaluate the cytoplasmic targeted delivery by using probe-conjugated nanoparticles in vivo. On the other hand, MMP probe was superior in specificity in vivo and tissue study. This comparative study will provide precise information about peptide-based optical probes, and allow their proper application to cancer diagnosis.

  20. Neuroprotective effect of synthetic chalcone derivatives as competitive dual inhibitors against μ-calpain and cathepsin B through the downregulation of tau phosphorylation and insoluble Aβ peptide formation.

    Science.gov (United States)

    Jeon, Kyung-Hwa; Lee, Eunyoung; Jun, Kyu-Yeon; Eom, Ji-Eun; Kwak, Soo Yeon; Na, Younghwa; Kwon, Youngjoo

    2016-10-01

    A series of chalcone derivatives were synthesized and evaluated for their μ-calpain and cathepsin B inhibitory activities. Among the tested chalcone derivatives, two compounds, 7 and 11, showed potent inhibitory activities against μ-calpain and cathepsin B and were selected for further evaluation. Compounds 7 and 11 showed enzyme inhibitory activities at the cellular level and displayed neuroprotective effects against oxidative stress-induced apoptosis in SH-SY5Y cells, a human neuroblastoma cell line. Moreover, compounds 7 and 11 reduced p25 formation, tau phosphorylation and insoluble Aβ peptide formation. Enzyme kinetic experiments and docking studies revealed that compounds 7 and 11 competitively inhibited both μ-calpain and cathepsin B enzymes. PMID:27318120

  1. Autophagy and cathepsin L are involved in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model

    Institute of Scientific and Technical Information of China (English)

    Wei-wei GU; Gui-zhen AO; Yong-ming ZHU; Shi-chang SUN; Qiang ZHOU; Jia-hong FAN; Katunuma NOBUHIKO

    2013-01-01

    Aim:2-(3',5'-Dimethoxybenzylidene) cyclopentanone (DMBC) is a novel synthetic compound with antinociceptive activities.The aim of this study was to investigate the roles of the autophagic-lysosomal pathway in the antinociceptive effect of DMBC in a mouse acetic acid-writhing model.Methods:Mouse acetic acid-writhing test and hotplate test were used to assess the antinociceptive effects of DMBC,3-MA (autophagy inhibitor) and Clik148 (cathepsin L inhibitor).The drugs were administered peripherally (ip) or centrally (icv).Results:Peripheral administration of 3-MA (7.5-30 mg/kg) or Clik148 (10-80 mg/kg) produced potent antinociceptive effect in acetic acid-writhing test.Central administration of 3-MA or Clik148 (12.5-50 nmol/L) produced comparable antinociceptive effect in acetic acid-writhing test.Peripheral administration of DMBC (25-50 mg/kg) produced potent antinociceptive effects in both acetic acidwrithing and hotplate tests.Furthermore,the antinociceptive effect produced by peripheral administration of DMBC (50 mg/kg) in acetic acid-writhing test was antagonized by low doses of 3-MA (3.75 mg/kg) or Clik148 (20 mg/kg) peripherally administered,but was not affected by 3-MA or Clik148 (25 nmol/L) centrally administered.Conclusion:Activation of central autophagy and cathepsin L is involved in nociception in mice,whereas peripheral autophagy and cathepsin L contributes,at least in part,to the antinociceptive effect of DMBC in mice.

  2. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

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    Iris J Gonzalez-Leal

    2014-09-01

    Full Text Available Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb and L (Ctsl play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC and macrophages (BMM from Ctsb-/- and Ctsl-/- mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb-/- BMDC express higher levels of MHC class II molecules than wild-type (WT and Ctsl-/- BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb-/- mice significantly up-regulated the levels of interleukin 12 (IL-12 expression, a key Th1-inducing cytokine. These findings indicate that Ctsb-/- BMDC display more pro-Th1 properties than their WT and Ctsl-/- counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.

  3. Cathepsin K null mice show reduced adiposity during the rapid accumulation of fat stores.

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    Marcella Funicello

    Full Text Available Growing evidences indicate that proteases are implicated in adipogenesis and in the onset of obesity. We previously reported that the cysteine protease cathepsin K (ctsk is overexpressed in the white adipose tissue (WAT of obese individuals. We herein characterized the WAT and the metabolic phenotype of ctsk deficient animals (ctsk-/-. When the growth rate of ctsk-/- was compared to that of the wild type animals (WT, we could establish a time window (5-8 weeks of age within which ctsk-/-display significantly lower body weight and WAT size as compared to WT. Such a difference was not observable in older mice. Upon treatment with high fat diet (HFD for 12 weeks ctsk-/- gained significantly less weight than WT and showed reduced brown adipose tissue, liver mass and a lower percentage of body fat. Plasma triglycerides, cholesterol and leptin were significantly lower in HFD-fed-ctsk-/- as compared to HFD-fed WT animals. Adipocyte lipolysis rates were increased in both young and HFD-fed-ctsk-/-, as compared to WT. Carnitine palmitoyl transferase-1 activity, was higher in mitochondria isolated from the WAT of HFD treated ctsk-/- as compared to WT. Together, these data indicate that ctsk ablation in mice results in reduced body fat content under conditions requiring a rapid accumulation of fat stores. This observation could be partly explained by an increased release and/or utilization of FFA and by an augmented ratio of lipolysis/lipogenesis. These results also demonstrate that under a HFD, ctsk deficiency confers a partial resistance to the development of dyslipidemia.

  4. Cathepsin B/cystatin C complex levels in sera from patients with lung and colorectal cancer.

    Science.gov (United States)

    Zore, I; Krasovec, M; Cimerman, N; Kuhelj, R; Werle, B; Nielsen, H J; Brünner, N; Kos, J

    2001-05-01

    A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 nM and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and B (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression. PMID:11517934

  5. Detection of esophageal squamous cell carcinoma by cathepsin B activity in nude mice.

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    Wei Ma

    Full Text Available BACKGROUND AND OBJECTIVE: Despite great progress in treatment, the prognosis for patients with esophageal squamous cell carcinoma (ESCC remains poor, highlighting the importance of early detection. Although upper endoscopy can be used for the screening of esophagus, it has limited sensitivity for early stage disease. Thus, development of new diagnosis approach to improve diagnostic capabilities for early detection of ESCC is an important need. The aim of this study was to assess the feasibility of using cathepsin B (CB as a novel imaging target for the detection of human ESCC by near-infrared optical imaging in nude mice. METHODS: Initially, we examined specimens from normal human esophageal tissue, intraepithelial neoplasia lesions, tumor in situ, ESCC and two cell lines including one human ESCC cell line (Eca-109 and one normal human esophageal epithelial cell line (HET-1A for CB expression by immunohistochemistry and western blot, respectively. Next, the ability of a novel CB activatable near-infrared fluorescence (NIRF probe detecting CB activity presented in Eca-109 cells was confirmed by immunocytochemistry. We also performed in vivo imaging of tumor bearing mice injected with the CB probe and ex vivo imaging of resected tumor xenografts and visceral organs using a living imaging system. Finally, the sources of fluorescence signals in tumor tissue and CB expression in visceral organs were identified by histology. RESULTS: CB was absent in normal human esophageal mucosa, but it was overexpressed in ESCC and its precursor lesions. The novel probe for CB activity specifically detected ESCC xenografts in vivo and in vitro. CONCLUSIONS: CB was highly upregulated in human ESCC and its precursor lesions. The elevated CB expression in ESCC allowed in vivo and in vitro detection of ESCC xenografts in nude mice. Our results support the usefulness of CB activity as a potential imaging target for the detection of human ESCC.

  6. Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis.

    Science.gov (United States)

    Nepal, Rajeev M; Mampe, Stephanie; Shaffer, Brian; Erickson, Ann H; Bryant, Paula

    2006-06-01

    Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading. PMID:16636015

  7. Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages.

    Science.gov (United States)

    Muschol, Nicole; Matzner, Ulrich; Tiede, Stephan; Gieselmann, Volkmar; Ullrich, Kurt; Braulke, Thomas

    2002-12-15

    The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of (125)I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [(35)S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [(33)P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed (33)P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [(35)S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain. PMID:12296771

  8. Cathepsin L in tumor angiogenesis and its therapeutic intervention by the small molecule inhibitor KGP94.

    Science.gov (United States)

    Sudhan, Dhivya R; Rabaglino, Maria B; Wood, Charles E; Siemann, Dietmar W

    2016-06-01

    A significant proportion of breast cancer patients harbor clinically undetectable micrometastases at the time of diagnosis. If left untreated, these micro-metastases may lead to disease relapse and possibly death. Hence, there is significant interest in the development of novel anti-metastatic agents that could also curb the growth of pre-established micrometastases. Like primary tumor, the growth of metastases also is driven by angiogenesis. Although the role of cysteine protease Cathepsin L (CTSL) in metastasis associated tumor cell functions such as migration and invasion is well recognized, its role in tumor angiogenesis remains less explored. The present study examines the contribution of CTSL to breast cancer angiogenesis and evaluates the anti-angiogenic efficacy of CTSL inhibitor KGP94. CTSL semi-quantitative RT-PCR analysis on breast tissue panels revealed significant upregulation of CTSL in breast cancer patients which strongly correlated with increased relapse and metastatic incidence and poor overall survival. Preclinically, CTSL ablation using shRNA or KGP94 treatment led to a significant reduction in MDA-MB-231 tumor cell induced angiogenesis in vivo. In-vitro assessments demonstrated a significant decrease in various angiogenic properties such as endothelial cell sprouting, migration, invasion, tube formation and proliferation in the presence of KGP94. Microarray analyses revealed a significant upregulation of cell cycle related genes by CTSL. Western blot analyses further confirmed upregulation of members of the cyclin family by CTSL. Collectively, these data indicate that CTSL is an important contributor to tumor angiogenesis and that the CTSL inhibition may have therapeutic utility in the treatment of breast cancer patients. PMID:27055649

  9. Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible.

    Science.gov (United States)

    Zhuo, Y; Gauthier, J-Y; Black, W C; Percival, M D; Duong, L T

    2014-10-01

    The cathepsin K (CatK) inhibitor odanacatib (ODN) is currently being developed for the treatment of osteoporosis. In clinical trials, efficacy and resolution of effect of ODN treatment on bone turnover biomarkers and accrued bone mass have been demonstrated. Here, we examine the effects of continuing treatment and discontinuation of ODN versus alendronate (ALN) on osteoclast (OC) function. First, accessibility and reversible engagement of active CatK in intracellular vesicles and resorption lacunae of actively resorbing OCs were demonstrated by the selective and reversible CatK inhibitors, BODIPY-L-226 (IC50=39nM) and L-873,724 (IC50=0.5nM). Next, mature human OCs on bone slices were treated with vehicle, ODN, or ALN for 2days, followed by either continuing with the same treatment, or replacement of the inhibitors by vehicle for additional times as specified per experimental conditions. Maintaining OCs on ODN or ALN significantly reduced CTx-I release compared to vehicle controls. However, only the treatment of OCs with ODN resulted in the formation of small shallow discrete resorption pits, retention of intracellular vesicles enriched with CatK and other lysosomal enzymes, increase in 1-CTP release and number of TRAP(+) OCs. Upon discontinuation of ODN treatment, OCs rapidly resumed bone resorption activity, as demonstrated by a return of OC functional markers (CTx-I, 1-CTP), cell number and size, morphology and number of resorption pits, and vesicular secretion of CatK toward the respective vehicle levels. As expected, discontinuation of ALN did not reverse the treatment-related inhibition of OC activity in the time frame of the experiment. In summary, this study demonstrated rapid kinetics of inhibition and reversibility of the effects of ODN on OC bone resorption, that differentiated the cellular mechanism of CatK inhibition from that of the bisphosphate antiresorptive ALN. PMID:25038310

  10. Cathepsin K Inhibition: A New Mechanism for the Treatment of Osteoporosis.

    Science.gov (United States)

    Duong, Le T; Leung, Albert T; Langdahl, Bente

    2016-04-01

    Cathepsin K (CatK), a cysteine protease, is highly expressed by osteoclasts and very efficiently degrades type I collagen, the major component of the organic bone matrix. Robust genetic and pharmacological preclinical studies consistently demonstrate that CatK inhibition increases bone mass, improves bone microarchitecture and strength. Recent advances in the understanding of the molecular and cellular mechanisms involved in bone modeling and remodeling suggest that inhibition of CatK decreases bone resorption, but increases the number of cells of osteoclast lineage. This in turn maintains the signals for bone formation, and perhaps may even increase bone formation on some cortical surfaces. Several CatK inhibitors, including relacatib, balicatib, odanacatib and ONO-5334 had entered clinical development for metabolic bone disorders with increased bone resorption, such as postmenopausal osteoporosis. However, odanacatib (ODN) is the only candidate continuing in development. ODN is a highly selective oral CatK inhibitor dosed once-weekly in humans. In a Phase 2 clinical trial, postmenopausal women treated with ODN had sustained reductions of bone resorption markers, while bone formation markers returned to normal after an initial decline within the first 2 years on treatment. In turn areal bone mineral density increased continuously at both spine and hip for up to 5 years. ODN has also been demonstrated to improve bone mass in women with postmenopausal osteoporosis previously treated with alendronate and in men with osteoporosis. ODN is currently in a worldwide Phase 3 fracture outcome trial for the treatment of postmenopausal osteoporosis with interim results supporting its anti-fracture efficacy at the spine, hip and non-vertebral sites. PMID:26335104

  11. Disposition and metabolism of the cathepsin K inhibitor odanacatib in humans.

    Science.gov (United States)

    Kassahun, Kelem; McIntosh, Ian; Koeplinger, Kenneth; Sun, Li; Talaty, Jennifer E; Miller, Deborah L; Dixon, Russell; Zajic, Stefan; Stoch, S Aubrey

    2014-05-01

    Odanacatib is a selective inhibitor of the cathepsin K enzyme that is expressed in osteoclasts involved in the degradation of bone organic matrix, and is being developed as a novel treatment of osteoporosis. Odanacatib has demonstrated increases in bone mineral density in postmenopausal women and is undergoing a pivotal phase III trial. The absorption, metabolism, and excretion of [(14)C]odanacatib were studied in healthy male volunteers (n = 6) after a single oral dose of 25 mg (100 µCi). Plasma, urine, and fecal samples were collected at intervals up to 34 days postdose. The pharmacokinetics of odanacatib were characterized by slow absorption (mean time to achieve maximum plasma concentration of 14.2 hours) and long apparent elimination half-life (mean t1/2 96.7 hours); 74.5% of the dose was recovered in feces and 16.9% in urine, resulting in a total recovery of 91.4%. Seven metabolites were identified in urine; the major pathway (methyl hydroxylation producing M8 and its derivatives) was largely dependent on CYP3A. Metabolites and odanacatib accounted for 77% and 23% of urinary radioactivity, respectively. In fecal extracts, the only radioactive components identified were odanacatib (60.9%) and M8 (9.5%). The fraction of odanacatib in feces derived from absorbed drug was estimated using a bioavailability value obtained from the results of a separate intravenous study. Collectively, the data indicate that odanacatib has a long t1/2 on account of its low metabolic intrinsic clearance, and that metabolism (principally mediated by CYP3A) and excretion of intact parent compound account for ∼70% and ∼30% of the clearance of odanacatib in humans. PMID:24553380

  12. Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications.

    Science.gov (United States)

    Kumar Vr, Santhosh; Darisipudi, Murthy N; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P; Mulay, Shrikant R; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D; Lindenmeyer, Maja T; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido; Anders, Hans-Joachim

    2016-06-01

    Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases. PMID:26567242

  13. THE ROLE OF CYTOKINE SYSTEM RANKL-RANK-OPG AND CATHEPSIN K IN THE PATHOGENESIS OF OSTEOPOROSIS: ACHIEVEMENTS AND PERSPECTIVES IN THE TREATMENT OF DISEASE

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    S. Sagalovsky

    2012-01-01

    Full Text Available The article presents review of literature dedicated to the contemporary view on the cellular-molecular mechanisms of the bone remodeling and pathogenesis of the osteoporosis. The discovery of the cytokine RANKL-RANK-OPG system and significant role of the cathepsin K in process bone remodeling has made progress in understanding the mechanisms development disease and possible to development drugs of the new generation – denosumab, a fully human RANKL monoclonal antibody and inhibitor cathepsin K odanacatib that inhibits of the bone resorption.

  14. Regulation of cathepsin G reduces the activation of proinsulin-reactive T cells from type 1 diabetes patients.

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    Fang Zou

    Full Text Available Autoantigenic peptides resulting from self-proteins such as proinsulin are important players in the development of type 1 diabetes mellitus (T1D. Self-proteins can be processed by cathepsins (Cats within endocytic compartments and loaded to major histocompatibility complex (MHC class II molecules for CD4(+ T cell inspection. However, the processing and presentation of proinsulin by antigen-presenting cells (APC in humans is only partially understood. Here we demonstrate that the processing of proinsulin by B cell or myeloid dendritic cell (mDC1-derived lysosomal cathepsins resulted in several proinsulin-derived intermediates. These intermediates were similar to those obtained using purified CatG and, to a lesser extent, CatD, S, and V in vitro. Some of these intermediates polarized T cell activation in peripheral blood mononuclear cells (PBMC from T1D patients indicative for naturally processed T cell epitopes. Furthermore, CatG activity was found to be elevated in PBMC from T1D patients and abrogation of CatG activity resulted in functional inhibition of proinsulin-reactive T cells. Our data suggested the notion that CatG plays a critical role in proinsulin processing and is important in the activation process of diabetogenic T cells.

  15. IGFBP-4 Anti-Angiogenic and Anti-Tumorigenic Effects Are Associated with Anti-Cathepsin B Activity

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    María J Moreno

    2013-05-01

    Full Text Available Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4 has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4, a region containing a thyroglobulin type 1 (Tg1 domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma.

  16. Differential expression of Cathepsin S and X in the spinal cord of a rat neuropathic pain model

    Directory of Open Access Journals (Sweden)

    Schmitz Beate

    2008-08-01

    Full Text Available Abstract Background Ample evidence suggests a substantial contribution of cellular and molecular changes in the spinal cord to the induction and persistence of chronic neuropathic pain conditions. While for a long time, proteases were mainly considered as protein degrading enzymes, they are now receiving growing interest as signalling molecules in the pain pathology. In the present study we focused on two cathepsins, CATS and CATX, and studied their spatiotemporal expression and activity during the development and progression of neuropathic pain in the CNS of the rat 5th lumbar spinal nerve transection model (L5T. Results Immediately after the lesion, both cathepsins, CATS and CATX, were upregulated in the spinal cord. Moreover, we succeeded in measuring the activity of CATX, which was substantially increased after L5T. The differential expression of these proteins exhibited the same spatial distribution and temporal progression in the spinal cord, progressing up to the medulla oblongata in the late phase of chronic pain. The cellular distribution of CATS and CATX was, however, considerably different. Conclusion The cellular distribution and the spatio-temporal development of the altered expression of CATS and CATX suggest that these proteins are important players in the spinal mechanisms involved in chronic pain induction and maintenance.

  17. Cathepsin D and MMP-9 activity increase following a high intensity exercise in hind limb muscles of young rats.

    Science.gov (United States)

    Carmeli, Eli; Haimovitz, Tal; Nemcovsky, E Carlos

    2007-01-01

    The influence of an intensive exercise regime on cathepsin D and MMP-9 activity in hind limb muscles was investigated. We hypothesized that high-intensity exercise would increase the number of these proteins, indicating their involvement in the pathogenesis of exercise-induced muscle injury. Muscle fibers from the gastrocnemius and soleus were used from young (6-mo-old) female rats (n = 6) who completed 10 consecutive days of treadmill running at high intensity (34 m min(-1) gradually up to 40 min per day), compared with nonrunning, age and sex-matched rats (n = 6). After a high-intensity exercise regime, cathepsin D activity significantly increased in the gastrocnemius (from 6.6 x 10(-3) to 10.7 x 10(-3) or 61% nM tyrosine x mg-1 protein x min-1) and the soleus (from 5.9 x 10(-3) to 8.9 x 10(-3) or 66%). The activity level of mRNA MMP-9, expressed as ng mg(-1) protein, increased in both muscles subjected to intensity running. The results of this study suggest that high-intensity running results in an elevation in the activity of lysosomal enzymes involved in matrix protein degradation. PMID:17569248

  18. IGFBP-4 Anti-Angiogenic and Anti-Tumorigenic Effects Are Associated with Anti-Cathepsin B Activity1

    Science.gov (United States)

    Moreno, María J; Ball, Marguerite; Rukhlova, Marina; Slinn, Jacqueline; L'Abbe, Denis; Iqbal, Umar; Monette, Robert; Hagedorn, Martin; O'Connor-McCourt, Maureen D; Durocher, Yves; Stanimirovic, Danica B

    2013-01-01

    Insulin-like growth factor-binding protein 4 (IGFBP-4/IBP-4) has potent IGF-independent anti-angiogenic and antitumorigenic effects. In this study, we demonstrated that these activities are located in the IGFBP-4 C-terminal protein fragment (CIBP-4), a region containing a thyroglobulin type 1 (Tg1) domain. Proteins bearing Tg1 domains have been shown to inhibit cathepsins, lysosomal enzymes involved in basement membrane degradation and implicated in tumor invasion and angiogenesis. In our studies, CIBP-4 was shown to internalize and co-localize with lysosomal-like structures in both endothelial cells (ECs) and glioblastoma U87MG cells. CIBP-4 also inhibited both growth factor-induced EC tubulogenesis in Matrigel and the concomitant increases in intracellular cathepsin B (CatB) activity. In vitro assays confirmed CIBP-4 capacity to block recombinant CatB activity. Biodistribution analysis of intravenously injected CIBP-4-Cy5.5 in a glioblastoma tumor xenograft model indicated targeted accumulation of CIBP-4 in tumors. Most importantly, CIBP-4 reduced tumor growth in this animal model by 60%. Pleiotropic anti-angiogenic and anti-tumorigenic activities of CIBP-4 most likely underlie its observed therapeutic potential against glioblastoma. PMID:23633927

  19. Up-regulation of microglial cathepsin C expression and activity in lipopolysaccharide-induced neuroinflammation

    Directory of Open Access Journals (Sweden)

    Fan Kai

    2012-05-01

    Full Text Available Abstract Background Cathepsin C (Cat C functions as a central coordinator for activation of many serine proteases in inflammatory cells. It has been recognized that Cat C is responsible for neutrophil recruitment and production of chemokines and cytokines in many inflammatory diseases. However, Cat C expression and its functional role in the brain under normal conditions or in neuroinflammatory processes remain unclear. Our previous study showed that Cat C promoted the progress of brain demyelination in cuprizone-treated mice. The present study further investigated the Cat C expression and activity in lipopolysaccharide (LPS-induced neuroinflammation in vivo and in vitro. Methods C57BL/6 J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg. Immunohistochemistry (IHC and in situ hybridization (ISH were used to analyze microglial activation, TNF-α, IL-1β, IL-6, iNOS mRNAs expressions and cellular localization of Cat C in the brain. Nitrite assay was used to examine microglial activation in vitro; RT-PCR and ELISA were used to determine the expression and release of Cat C. Cat C activity was analyzed by cellular Cat C assay kit. Data were evaluated for statistical significance with paired t test. Results Cat C was predominantly expressed in hippocampal CA2 neurons in C57BL/6 J mice under normal conditions. Six hours after LPS injection, Cat C expression was detected in cerebral cortical neurons; whereas, twenty-four hours later, Cat C expression was captured in activated microglial cells throughout the entire brain. The duration of induced Cat C expression in neurons and in microglial cells was ten days and three days, respectively. In vitro, LPS, IL-1β and IL-6 treatments increased microglial Cat C expression in a dose-dependent manner and upregulated Cat C secretion and its activity. Conclusions Taken together, these data indicate that LPS and proinflammatory cytokines IL-1β, IL-6 induce the

  20. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice.

    Science.gov (United States)

    Kueakhai, Pornanan; Changklungmoa, Narin; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Itagaki, Tadashi; Sobhon, Prasert

    2015-10-01

    In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that r

  1. Cathepsins B1 and B2 in the neuropathogenic schistosome Trichobilharzia regenti: distinct gene expression profiles and presumptive roles throughout the life cycle

    Czech Academy of Sciences Publication Activity Database

    Dolečková, K.; Albrecht, Tomáš; Mikeš, L.; Horák, P.

    2010-01-01

    Roč. 107, č. 3 (2010), s. 751-755. ISSN 0932-0113 R&D Projects: GA MŠk LC06073 Institutional research plan: CEZ:AV0Z60930519 Keywords : Cathepsin * Schistosome * Trichobilharzia * Helminth * Neuropathogen Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 1.812, year: 2010

  2. 4-tert-Octylphenol stimulates the expression of cathepsins in human breast cancer cells and xenografted breast tumors of a mouse model via an estrogen receptor-mediated signaling pathway

    International Nuclear Information System (INIS)

    Highlights: ► Cathepsins B and D were markedly enhanced by octylphenol (OP) in MCF-7 cells. ► OP may accelerate breast cancer cell growth and cathepsins via ER-mediated signaling. ► Breast cancer cells exposed with OP to mouse model were more aggressive. ► OP can promote metastasis through the amplification of cathepsins B and D via ER-mediated signaling pathway. -- Abstract: Endocrine disrupting chemicals (EDCs) are defined as environmental compounds that modulate steroid hormone receptor-dependent responses an abnormal manner, resulting in adverse health problems for humans such as cancer growth and metastasis. Cathepsins are proteases that have been implicated in cancer progression. However, there have been few studies about the association between cathepsins and estrogenic chemicals during the cancer progression. In this study, we examined the effect(s) of 4-tert-octylphenol (OP), a potent EDC, on the expression of cathepsins B and D in human MCF-7 breast cancer cells and a xenograft mouse model. Treatment with OP significantly induced the proliferation MCF-7 cells in an MTT assay. In addition, the expression of cathepsins B and D was markedly enhanced in MCF-7 cells at both the transcriptional and the translational levels following treatment with E2 or OP up to 48 h. These results demonstrated the ability of OP to disrupt normal transcriptional regulation of cathepsins B and D in human breast cancer cells. However, the effects of OP on cell growth or overexpression of cathepsins by inhibiting ER-mediated signaling were abolished by an ER antagonist and siRNA specific for ERα. In conclusion, our findings suggest that OP at 10−6 M, like E2, may accelerate breast cancer cell proliferation and the expression of cathepsins through an ER-mediated signaling pathway. In addition, the breast cancer cells exposed with OP to a xenograft mouse model were more aggressive according to our histological analysis and showed markedly increased expression of cathepsin

  3. Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

    International Nuclear Information System (INIS)

    Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG

  4. Amyloid β oligomers induce interleukin-1β production in primary microglia in a cathepsin B- and reactive oxygen species-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Taneo, Jun; Adachi, Takumi [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Yoshida, Aiko; Takayasu, Kunio [Responses to Environmental Signals and Stresses, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto, Kyoto 606-8501 (Japan); Takahara, Kazuhiko, E-mail: ktakahar@zoo.zool.kyoto-u.ac.jp [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan); Inaba, Kayo [Department of Animal Development and Physiology, Kyoto University, Yoshida-Konoe, Sakyo, Kyoto 606-8501 (Japan); Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Tokyo 102-0081 (Japan)

    2015-03-13

    Amyloid β (Aβ) peptide, a causative agent of Alzheimer's disease, forms two types of aggregates: oligomers and fibrils. These aggregates induce inflammatory responses, such as interleukin-1β (IL-1β) production by microglia, which are macrophage-like cells located in the brain. In this study, we examined the effect of the two forms of Aβ aggregates on IL-1β production in mouse primary microglia. We prepared Aβ oligomer and fibril from Aβ (1–42) peptide in vitro. We analyzed the characteristics of these oligomers and fibrils by electrophoresis and atomic force microscopy. Interestingly, Aβ oligomers but not Aβ monomers or fibrils induced robust IL-1β production in the presence of lipopolysaccharide. Moreover, Aβ oligomers induced endo/phagolysosome rupture, which released cathepsin B into the cytoplasm. Aβ oligomer-induced IL-1β production was inhibited not only by the cathepsin B inhibitor CA-074-Me but also by the reactive oxygen species (ROS) inhibitor N-acetylcysteine. Random chemical crosslinking abolished the ability of the oligomers to induce IL-1β. Thus, multimerization and fibrillization causes Aβ oligomers to lose the ability to induce IL-1β. These results indicate that Aβ oligomers, but not fibrils, induce IL-1β production in primary microglia in a cathepsin B- and ROS-dependent manner. - Highlights: • We prepared amyloid β (Aβ) fibrils with minimum contamination of Aβ oligomers. • Primary microglia (MG) produced IL-1β in response to Aβ oligomers, but not fibrils. • Only Aβ oligomers induced leakage of cathepsin B from endo/phagolysosomes. • IL-1β production in response to Aβ oligomers depended on both cathepsin B and ROS. • Crosslinking reduced the ability of the Aβ oligomers to induce IL-1β from MG.

  5. Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes

  6. Cathepsin L of the sea cucumber Apostichopus japonicus-molecular characterization and transcriptional response to Vibrio splendidus infection.

    Science.gov (United States)

    Yang, Jingwen; Liu, Huihui; Zheng, Gang; Xiang, Xiaowei; Lv, Zhenming; Wang, Tianming

    2016-02-01

    Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response. PMID:26777896

  7. Frequency of CFTR, SPINK1, and Cathepsin B Gene Mutation in North Indian Population: Connections between Genetics and Clinical Data

    Directory of Open Access Journals (Sweden)

    Shweta Singh

    2014-01-01

    Full Text Available Objectives. Genetic mutations and polymorphisms have been correlated with chronic pancreatitis (CP. This study aims to investigate the association of genetic variants of cystic fibrosis transmembrane conductance regulator (CFTR and serine protease inhibitor Kazal type 1 (SPINK-1 genes and Cathepsin B gene polymorphisms with CP and to associate genetic backgrounds with clinical phenotypes. Methods. 150 CP patients and 150 normal controls were enrolled consecutively. We analyzed SPINK-1 N34S and IVS3+2T>C gene mutations by PCR-restriction-fragment length polymorphism (RFLP. The identification of DF508, G551D, G542X, R117H, and W1282X mutations was carried out by ARMS-PCR. S549N mutation, IVS8 polyTn polymorphism, and Cathepsin B Lec26Val were analysed by PCR-RFLP, nested PCR, and PCR-RFLP plus sequencing, respectively. Results. We found a significant association of SPINK1 (N34S gene polymorphism. IVS1−37T>C polymorphism shows linkage with 101A>G. 300 chromosomes belonging to the CFTR subgroup exhibited minor allele frequency of 0.04, 0.03, 0.03, 0.013, 0.006, and 0.02 for DF508, G452X, G551D, S549N, R117H, and IVS8 T5, respectively. Except for R117H and IVS8 T5 polymorphisms, all other mutations showed significant variation. Conclusion. Analysis of potential susceptibility variants is needed to support nature of the genes and environment in pancreatitis. This data may help establish genetic screening and prenatal setup for Indian population.

  8. Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

    Science.gov (United States)

    2013-01-01

    Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. Results The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R↓A:A↓K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. Conclusions E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident. PMID:24341625

  9. Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.

    Directory of Open Access Journals (Sweden)

    Ramarao Malla

    Full Text Available BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

  10. Enhanced Autophagy and Reduced Expression of Cathepsin D Are Related to Autophagic Cell Death in Epstein-Barr Virus-Associated Nasal Natural Killer/T-Cell Lymphomas: An Immunohistochemical Analysis of Beclin-1, LC3, Mitochondria (AE-1), and Cathepsin D in Nasopharyngeal Lymphomas

    International Nuclear Information System (INIS)

    This study investigated autophagy in 37 cases of nasopharyngeal lymphomas including 23 nasal natural killer (NK)/T-cell lymphomas (NKTCL), 3 cytotoxic T-cell lymphomas (cytotoxic-TML) and 9 B-cell lymphomas (BML) by means of antigen-retrieval immunohistochemistry of beclin-1, LC3, mitochondria (AE-1) and cathepsin D. Peculiar necrosis was noted in EBV+ lymphomas comprising 21 NKTCL, 2 cytotoxic-TML and 1 BML. Lymphomas without peculiar necrosis showed high expression of beclin-1, macrogranular cytoplasmal stain of LC3 with sporadic nuclear stain, a hallmark of autophagic cell death (ACD), some aggregated mitochondria and high expression of cathepsin D, suggesting a state of growth with enhanced autophagy with sporadic ACD. EBV+ NKTCL with the peculiar necrosis, showed significantly low level of macrogranular staining of LC3, aggregated mitochondria and low expression of cathepsin D in the cellular areas when degenerative lymphoma cells showed decreased beclin-1, significantly advanced LC3-labeled autophagy, residual aggregated mitochondria and significantly reduced expression of cathepsin D, suggesting advanced autophagy with regional ACD. Consequently it was suggested that enhanced autophagy and reduced expression of lysosomal enzymes induced regional ACD under EBV infection in NKTCL

  11. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone;

    2004-01-01

    Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, z......VAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently...... been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes...

  12. Progress on Action of Cathepsin K in Bone Resorption%组织蛋白酶K在骨吸收中的作用研究进展

    Institute of Scientific and Technical Information of China (English)

    王东; 顾建红; 刘宗平

    2014-01-01

    组织蛋白酶 K(Cathepsin K,CK)是一种溶酶体半胱氨酸蛋白酶,属于番木瓜蛋白酶超家族。在破骨细胞(osteoclast,OC)介导的骨吸收过程中有效降解骨组织中的基质蛋白,在骨吸收过程中发挥重要作用。研究 CK 在骨吸收中的重要性,为治疗骨代谢疾病开辟了新的途径。论文就组织蛋白酶 K 的结构和功能及在骨吸收过程中与破骨细胞的相互作用和表达水平的调控机制进行了阐述。%Cathepsin K is one of lysosomal cysteine proteases,belongs to the papaya protease superfamily,highly expressed in the process of bone resorption mediated by osteoclasts and degrade matrix proteins in bone tissue ef-fectively,plays a significant role in the process on bone resorption.Study of the importance of cathepsin k in bone resorption opened up a new approach to metabolic bone disease treatment.In this mini-review,we mainly focused on the structure and function of cathepsin K and the interaction with osteoclast,the regulation mechanisms of ex-pression levels in the process of bone resorption.

  13. Mapping the Pro-Peptide of the Schistosoma mansoni Cathepsin B1 Drug Target: Modulation of Inhibition by Heparin and Design of Mimetic Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Jílková, Adéla; Vondrášek, Jiří; Marešová, Lucie; Caffrey, C. R.; Mareš, Michael

    2011-01-01

    Roč. 6, č. 6 (2011), s. 609-617. ISSN 1554-8929 R&D Projects: GA ČR GA203/09/1585; GA AV ČR KJB400550516; GA AV ČR IAA400550705 Grant ostatní: NATO(XE) NATO LST/CLG 980187 Institutional research plan: CEZ:AV0Z40550506 Keywords : Schistosoma mansoni * cathepsin B * propeptide Subject RIV: CE - Biochemistry Impact factor: 6.446, year: 2011

  14. Peri-implant crevicular fluid levels of cathepsin-K, RANKL, and OPG around standard, short, and mini dental implants after prosthodontic loading

    OpenAIRE

    Alan, Raif; Marakoğlu, İsmail; Haliloğlu, Seyfullah

    2015-01-01

    Purpose Despite the high success rates of endosseous dental implants, their placement is restricted according to the height and volume of bone available. The use of short or mini dental implants could be one way to overcome this limitation. Thus, this study aimed to compare standard, short, and mini dental implants with regard to associated clinical parameters and peri-implant crevicular fluid (PICF) levels of cathepsin -K (CTSK), RANK ligand (RANKL), and osteoprotegerin (OPG), after prosthod...

  15. The Influence of Linker Length on the Properties of Cathepsin S Cleavable 177Lu-labeled HPMA Copolymers for Pancreatic Cancer Imaging

    OpenAIRE

    Shi, Wen; Wagh, Nilesh K.; Zhou, Zhengyuan; Jia, Yinnong; Brusnahan, Susan K.; Garrison, Jered C.

    2014-01-01

    N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymers have shown promise for application in the detection and staging of cancer. However, non-target accumulation, particularly in the liver and spleen, hinders the detection of resident or nearby metastatic lesions thereby decreasing diagnostic effectiveness. Our laboratory has pursued the development of cathepsin S susceptible linkers (CSLs) to reduce the non-target accumulation of diagnostic/radiotherapeutic HPMA copolymers. In this study, we ...

  16. Peroxisome Proliferator-activated Receptor γ Induces Apoptosis and Inhibits Autophagy of Human Monocyte-derived Macrophages via Induction of Cathepsin L: POTENTIAL ROLE IN ATHEROSCLEROSIS*

    OpenAIRE

    Mahmood, Dler Faieeq Darweesh; Jguirim-Souissi, Imene; Khadija, El-Hadri; Blondeau, Nicolas; Diderot, Vimala; Amrani, Souliman; Slimane, Mohamed-Naceur; Syrovets, Tatiana; Simmet, Thomas; Rouis, Mustapha

    2011-01-01

    Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) γ, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPARγ might affect macrophage functi...

  17. Long-term effects of recurrent neonatal seizures on neurobehavioral function and related gene expression and its intervention by inhibitor of cathepsin B.

    Science.gov (United States)

    Ni, Hong; Yan, Jian-Zhen; Zhang, Le-Ling; Feng, Xing; Wu, Xi-Ru

    2012-01-01

    Cathepsins are families of proteases that have been reported to play the key roles in neuroexcitotoxicity. The present study was sought to determine the effect of CBI, a cathepsin B inhibitor, in the prevention of neurobehavioral deficits after inhalant flurothyl-induced recurrent neonatal seizures in rats. We examined the expression pattern of autophagy-related genes at acute phase after the last seizures using western blot method, and evaluated behavioral deficits during postnatal day 28 (P28) to P35. The results showed improved neurological scores and learning ability in CBI-treated rats compared with the nontreated control. Flurothyl-induced increases in the ratio of LC3-II/LC3-I, Beclin-1 and Cathepsin-B were blocked by pre-treatment with CBI at 1.5, 3, 6 and 24 h after the last seizures in hippocampus and cerebral cortex by western blot analysis. Meanwhile, CBI also reversed flurothyl-induced down-regulation of Bcl-2 protein levels. Furthermore, in the long-term time point of 35 days (P35), PRG-1 mRNA and protein level in hippocampus and cerebral cortex of recurrent seizure group were up-regulated when compared to the control rats; meanwhile, the up-regulated expression of PRG-1 were robustly inhibited by CBI. These date demonstrated, for the first time, that lysosomal enzymes participate in neonatal seizure-induced brain damage and that modulation of cathepsin B may offer a new strategy for the development of therapeutic interventions for treatment of developmental seizure-induced brain damage. PMID:21842268

  18. A dot-ELISA using a partially purified cathepsin-L-like protein fraction from Taenia solium cysticerci, for the diagnosis of human neurocysticercosis

    OpenAIRE

    Piña, R; Gutiérrez, A H; Gilman, R. H.; Rueda, D, R.; Sifuentes, C; Flores, M.; Sheen, P; Rodriguez, S.; GarcÍa, H H; Zimic, M

    2011-01-01

    Human neurocysticercosis (NCC), caused by the cestode Taenia solium, is responsible for a significant amount of neurological morbidity and epilepsy in developing countries. The disease remains highly endemic in many areas, despite several efforts and interventions to control it. A simple, cheap and fast diagnostic assay that is suitable for use in field conditions is highly desired. In immunodiagnostics based on western immunoblots or standard ELISA, a cathepsin-L-like protein purified from t...

  19. The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

    Directory of Open Access Journals (Sweden)

    Violeta Morin

    Full Text Available Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

  20. Polymorphisms of the porcine cathepsins, growth hormone-releasing hormone and leptin receptor genes and their association with meat quality traits in Ukrainian Large White breed.

    Science.gov (United States)

    Balatsky, Viktor; Bankovska, Irina; Pena, Ramona N; Saienko, Artem; Buslyk, Tetyana; Korinnyi, Sergii; Doran, Olena

    2016-06-01

    Cathepsins, growth hormone-releasing hormone (GHRH) and leptin receptor (LEPR) genes have been receiving increasing attention as potential markers for meat quality and pig performance traits. This study investigated the allele variants in four cathepsin genes (CTSB, CTSK, CTSL, CTSS), GHRH and LEPR in pure-bred Ukrainian Large White pigs and evaluated effects of the allele variants on meat quality characteristics. The study was conducted on 72 pigs. Genotyping was performed using PCR-RFLP technique. Meat quality characteristics analysed were intramuscular fat content, tenderness, total water content, ultimate pH, crude protein and ashes. A medium level of heterozygosity values was established for GHRH and LEPR genes which corresponded to very high levels of informativeness indexes. Cathepsins CTSL, CTSB and CTSK had a low level of heterozygosity, and CTSS did not segregate in this breed. Association studies established that intramuscular fat content and tenderness were affected by the allele variance in GHRH and LEPR but not by CTSB and CTSL genes. The GHRH results could be particularly relevant for the production of lean prime cuts as the A allele is associated with both, a lower meat fat content and better tenderness values, which are two attributes highly regarded by consumers. Results of this study suggest that selective breeding towards GHRH/AA genotype would be particularly useful for improving meat quality characteristics in the production systems involving lean Large White lines, which typically have less than 2 % intramuscular fat content. PMID:27075656

  1. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

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    Kathleen M Averette

    Full Text Available NOD-like receptors (NLRs are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT, is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP. The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

  2. Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

    Institute of Scientific and Technical Information of China (English)

    Tadeusz Sebzda; Piotr Hanczyc; Yousif Saleh; Bernice F Akinpelumi; Maciej Siewinski; Jerzy Rudnicki

    2005-01-01

    AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Norris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Norris 5123 hepatoma.METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time,tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates.RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls.The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e.7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment (P<0.0001).CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

  3. Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis

    Directory of Open Access Journals (Sweden)

    Zhou Chenhui

    2011-07-01

    Full Text Available Abstract Background Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB and to investigate its diagnostic value for human helminthiases. Results The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases. Conclusions Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.

  4. Azilsartan increases levels of IL-10, down-regulates MMP-2, MMP-9, RANKL/RANK, Cathepsin K and up-regulates OPG in an experimental periodontitis model.

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    Aurigena Antunes de Araújo

    Full Text Available AIMS: The aim of this study was to evaluate the effects of azilsartan (AZT on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs, receptor activator of nuclear factor κB ligand (RANKL, receptor activator of nuclear factor κB (RANK, osteoprotegerin (OPG, cyclooxygenase-2 (COX-2, and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into 5 groups of 10 rats each: (1 nonligated, water; (2 ligated, water; (3 ligated, 1 mg/kg AZT; (4 ligated, 5 mg/kg AZT; and (5 ligated, 10 mg/kg AZT. All groups were treated with saline or AZT for 10 days. Periodontal tissues were analyzed by histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1β, IL-10, TNF-α, myeloperoxidase (MPO, and glutathione (GSH were determined by ELISA. RESULTS: Treatment with 5 mg/kg AZT resulted in reduced MPO (p<0.05 and IL-1β (p<0.05, increased levels of IL-10 (p<0.05, and reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and increased expression of OPG. CONCLUSIONS: These findings reveal that AZT increases anti-inflammatory cytokines and GSH and decreases bone loss in ligature-induced periodontitis in rats.

  5. The importance of pH in regulating the function of the Fasciola hepatica cathepsin L1 cysteine protease.

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    Jonathan Lowther

    Full Text Available The helminth parasite Fasciola hepatica secretes cathepsin L cysteine proteases to invade its host, migrate through tissues and digest haemoglobin, its main source of amino acids. Here we investigated the importance of pH in regulating the activity and functions of the major cathepsin L protease FheCL1. The slightly acidic pH of the parasite gut facilitates the auto-catalytic activation of FheCL1 from its inactive proFheCL1 zymogen; this process was approximately 40-fold faster at pH 4.5 than at pH 7.0. Active mature FheCL1 is very stable at acidic and neutral conditions (the enzyme retained approximately 45% activity when incubated at 37 degrees C and pH 4.5 for 10 days and displayed a broad pH range for activity peptide substrates and the protein ovalbumin, peaking between pH 5.5 and pH 7.0. This pH profile likely reflects the need for FheCL1 to function both in the parasite gut and in the host tissues. FheCL1, however, could not cleave its natural substrate Hb in the pH range pH 5.5 and pH 7.0; digestion occurred only at pH

  6. Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

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    Mark W Robinson

    Full Text Available BACKGROUND: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3, liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains. Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. CONCLUSIONS/SIGNIFICANCE: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.

  7. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    Science.gov (United States)

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. PMID:26884336

  8. Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity.

    Science.gov (United States)

    Lewis, Patrick A; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R; Collinge, John; Jackson, Graham S

    2006-04-15

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity. PMID:16441239

  9. Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

    Science.gov (United States)

    Lewis, Patrick A.; Properzi, Francesca; Prodromidou, Kanella; Clarke, Anthony R.; Collinge, John; Jackson, Graham S.

    2006-01-01

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrPC. Removal of the GPI anchor from PrPSc requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrPSc in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrPSc, (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrPSc or on prion infectivity. PMID:16441239

  10. Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

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    Kristine Porter

    Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  11. THE PROGNOSIS SIGNIFICANCE OF CATHEPSIN-D EXPRESSION IN THE DIFFERENT LOCATIONS IN AXILLARY NODES NEGATIVE CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: The aim of this study was to investigate Cathepsin-D (Cath-D) expression in different location and its relationship with prognosis in the axillary lymph nodes negative (ANN) breast cancer patients. Methods: Cath-D expression in 192 cases of breast carcinoma were examined by immunohistochemistry. Depending on different parts of expression, three evaluating methods were used, compared and analysed. Results: The positive rate of Cath-D expression in ANN breast cancer with poor prognosis group and axillary nodes positive (ANP) group were significantly higher than that in ANN breast cancer with good prognosis group (x2=23.20, P0.05). Cath-D expression in stromal cells had no statistical difference among the three groups (x2=1.56, P>0.05). When the Cath-D expression in cancer and stromal cells were counted into the positive rate, it was near the same (u1=0.47, u2=1.41, P>0.05). Conclusion: These results suggest that Cath-D expression is one of the powerful prognostic markers in ANN breast cancer. It's a reliable, practical, and convenient method to observe and evaluate Cath-D expression in cancer cells.

  12. Chemoproteomic Strategy to Quantitatively Monitor Transnitrosation Uncovers Functionally Relevant S-Nitrosation Sites on Cathepsin D and HADH2.

    Science.gov (United States)

    Zhou, Yani; Wynia-Smith, Sarah L; Couvertier, Shalise M; Kalous, Kelsey S; Marletta, Michael A; Smith, Brian C; Weerapana, Eranthie

    2016-06-23

    S-Nitrosoglutathione (GSNO) is an endogenous transnitrosation donor involved in S-nitrosation of a variety of cellular proteins, thereby regulating diverse protein functions. Quantitative proteomic methods are necessary to establish which cysteine residues are most sensitive to GSNO-mediated transnitrosation. Here, a competitive cysteine-reactivity profiling strategy was implemented to quantitatively measure the sensitivity of >600 cysteine residues to transnitrosation by GSNO. This platform identified a subset of cysteine residues with a high propensity for GSNO-mediated transnitrosation. Functional characterization of previously unannotated S-nitrosation sites revealed that S-nitrosation of a cysteine residue distal to the 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2) active site impaired catalytic activity. Similarly, S-nitrosation of a non-catalytic cysteine residue in the lysosomal aspartyl protease cathepsin D (CTSD) inhibited proteolytic activation. Together, these studies revealed two previously uncharacterized cysteine residues that regulate protein function, and established a chemical-proteomic platform with capabilities to determine substrate specificity of other cellular transnitrosation agents. PMID:27291402

  13. Design, Synthesis, and Biological Evaluation of New Cathepsin B-Sensitive Camptothecin Nanoparticles Equipped with a Novel Multifuctional Linker.

    Science.gov (United States)

    Zhang, Xuan; Tang, Kaiyong; Wang, Hong; Liu, Yaqian; Bao, Bin; Fang, Yanfen; Zhang, Xiongwen; Lu, Wei

    2016-05-18

    Traditional antitumor drugs such as camptothecin and paclitaxel derivatives are widely used in cancer chemotherapy. However, the major defects of those agents include severe toxicity and poor water solubility. With these in mind, a novel multifunctional linker was designed and two Cathepsin B (CTB) sensitive CPT conjugates (9a and 9b) were synthesized. Through click chemistry, additional functional group mPEG2000 can be easily introduced into these conjugates. The introduction of mPEG2000 fragment resulted in the formation of nanoparticles 1a and 1b (average particle sizes were 216.9 and 257.9 nm, respectively) with significantly increased water solubility (more than 19 000-fold). The release of therapeutic drug SN-38 in the presence of CTB was confirmed by HPLC and prodrug 1a showed potent in vitro cytotoxicity against all tested cell lines. Impressively, compared with irinotecan, CTB sensitive prodrug 1a displayed similar in vivo efficacy with remarkable decreased in vivo toxicity. PMID:27070848

  14. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    Science.gov (United States)

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis. PMID:25274570

  15. Cathepsin B, cathepsin L, and cystatin C in the porcine uterus and placenta: potential roles in endometrial/placental remodeling and in fluid-phase transport of proteins secreted by uterine epithelia across placental areolae.

    Science.gov (United States)

    Song, Gwonhwa; Bailey, Daniel W; Dunlap, Kathrin A; Burghardt, Robert C; Spencer, Thomas E; Bazer, Fuller W; Johnson, Greg A

    2010-05-01

    Cathepsins (CTSB and CTSL1) and their inhibitor, cystatin C (CST3), remodel uterine endometrium and placenta for transport of gases, micronutrients, and macromolecules essential for development and growth of the conceptus (embryo/fetus and placental membranes). We examined the temporal/spatial control of expression for CTSB, CTSL1, and CST3 mRNAs in endometria and placentae of pigs using three developmental models: 1) pigs were hysterectomized during the estrous cycle or pregnancy; 2) cyclic pigs were injected with estrogen to induce pseudopregnancy and were hysterectomized; and 3) pigs were ovariectomized, injected with progesterone, and hysterectomized. The abundance of CTSB, CTSL1, and CST3 mRNAs increased in endometrial epithelia during pregnancy and in response to exogenous progesterone but not estrogen. CST3 was also expressed in cells scattered within the stratum compactum stroma. Progesterone decreased epithelial but increased stromal compartment expression of CST3. CTSB increased in all chorionic epithelia, but CTSL1 was limited to chorionic epithelia that form areolae to absorb secretions from uterine glands. Based on the placental and endometrial distribution of CTSL1, we examined expression in the neonatal enterocytes known to transport immunoglobulins from colostrum. CTSL1 was also expressed in enterocytes of intestine from neonatal piglets. Therefore, CTSL1 is expressed by endometrial epithelia, placental areolae, and neonatal intestine, and it may function in the transport of macromolecules across these epithelia. Our results support the idea that reciprocal interactions between CSTL1, CTSB, and CST3 may be required to remodel endometrial and placental tissues for close apposition between maternal and fetal vasculatures and to facilitate transplacental transport of gases, micronutrients (amino acids, glucose), and macromolecules (proteins). Cysteine proteases and their inhibitors may also specifically modify proteins for successful utilization and

  16. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer

    International Nuclear Information System (INIS)

    Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging. The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%), CATB (82%), uPA (69%), CATL (41%) were higher than those of CEA or CA 19-9 (30% and 18%, respectively). PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%). High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004), CATL (P = 0.02), PAI-1 (P = 0.01) and CA 19-9 (P = 0.004) had a significant prognostic impact. PAI-1 (P = 0.001), CATB (P = 0.04) and CA 19-9 (P = 0.02) proved as independent prognostic variables. At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer

  17. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

    Science.gov (United States)

    Ferrara, Taíse Fernanda da Silva; Schneider, Vanessa Karine; Kishi, Luciano Takeshi; Carmona, Adriana Karaoglanovic; Alves, Marcio Fernando Madureira; Belasque-Júnior, Jose; Rosa, José César; Hunter, Wayne Brian; Henrique-Silva, Flávio; Soares-Costa, Andrea

    2015-01-01

    Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control. PMID:26717484

  18. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae: A Putative Target for Control of Citrus Huanglongbing.

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    Taíse Fernanda da Silva Ferrara

    Full Text Available Huanglonbing (HLB is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB. DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM. The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM and CaneCPI-4 (Ki = 0.05 nM and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM. RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  19. Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner.

    Science.gov (United States)

    Rasid, Orhan; Mériaux, Véronique; Khan, Erin M; Borde, Chloé; Ciulean, Ioana S; Fitting, Catherine; Manoury, Bénédicte; Cavaillon, Jean-Marc; Doyen, Noëlle

    2016-05-01

    A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection. PMID:27182703

  20. Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner

    Science.gov (United States)

    Mériaux, Véronique; Khan, Erin M.; Borde, Chloé; Ciulean, Ioana S.; Fitting, Catherine; Manoury, Bénédicte; Cavaillon, Jean-Marc; Doyen, Noëlle

    2016-01-01

    A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection. PMID:27182703

  1. The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA using recombinant cathepsin L protease.

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    Bibiana Gonzales Santana

    Full Text Available BACKGROUND: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. METHODOLOGY/PRINCIPAL FINDINGS: Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. CONCLUSIONS/SIGNIFICANCE: A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in

  2. Identification of novel mutation in cathepsin C gene causing Papillon-Lefèvre Syndrome in Mexican patients

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    Romero-Quintana José G

    2013-01-01

    Full Text Available Abstract Background Papillon-Lefèvre Syndrome (PLS is a type IV genodermatosis caused by mutations in cathepsin C (CTSC, with a worldwide prevalence of 1–4 cases per million in the general population. In México, the prevalence of this syndrome is unknown, and there are few case reports. The diagnosis of twenty patients in the state of Sinaloa highlights the need to characterize this syndrome in Mexicans. Methods To understand the basis of PLS in Mexicans, the gene expression, enzymatic activity and mutational analysis of CTSC were assayed in nine PLS patients and their relatives. Frequencies of CTSC gene polymorphisms and HLA alleles were determined in these patients, their relatives, and the population. Results Patients showed normal CTSC gene expression, but a deep reduction (up to 85% in enzymatic activity in comparison to unrelated healthy individuals. A novel loss-of-function mutation, c.203 T >; G (p.Leu68Arg, was found in all patients, and some carried the polymorphism c.458C >; T (p.Thr153Ile. Allelic frequencies in patients, relatives and controls were 88.89%, 38.24% and 0.25% for G (c.203 T >; G; and 11.11%, 8.82% and 9.00% for T (c.458C >; T. HLA-DRB1*11 was found significantly more frequent (P = 0.0071 in patients than controls (33.33% vs. 7.32%, with an estimated relative risk of 6.33. Conclusions The novel loss-of function mutation of CTSC gene (c.203 T >; G found in patients correlated with their diminished enzymatic activity, and HLA-DRB1*11 was found to be associated with PLS. The study of more PLS patients may give more insights into the etiology of the disease as well as its prevalence in México.

  3. Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

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    Gao-Feng Qiu

    2009-01-01

    Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

  4. Increased expression of cathepsin L: a novel independent prognostic marker of worse outcome in hepatocellular carcinoma patients.

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    Jian Ruan

    Full Text Available OBJECTIVES: To investigate the expression and role of Cathepsin L (CTSL in Hepatocellular carcinoma (HCC tissue and cell line (MHCC-97H, and to evaluate the clinical and prognostic significance of CTSL protein in patients with HCC. METHODS: The expression of CTSL was examined in HCC tissue and MHCC-97H cells by Western-blotting, Real-time PCR and immunohistochemical staining. Cell growth curve assay and colony formation assay were used to verify the effect of CTSL on the proliferation and tumor progression ability of MHCC-97H cells. Tumor formation assay in nude mice was used to analyze the effect of CTSL on the tumorigenicity of MHCC-97H cells. RESULTS: The status of CTSL protein in carcinoma tissues is much higher than that in paracarcinoma tissues. The overall survival of the patients with high CTSL expression was significantly shorter than the low CTSL expression group. high CTSL expression was significantly correlated with advanced clinical staging, histological grade and tumor recurrence. In vitro experiments demonstrated that over-expression of CTSL in MHCC-97H cells promoted cell proliferation and tumor progression ability. Down-regulation of CTSL showed the opposite effects. Over-expression of CTSL increase the tumorigenicity of MHCC-97H cells by in vivo experiments. Moreover, multivariate analysis suggested that CTSL expression might be an independent prognostic indicator for the survival of HCC patients after curative surgery. CONCLUSIONS: CTSL might involve in the development and progression of HCC as a oncogene, and thereby may be a valuable prognostic marker for HCC patients.

  5. Novel inhibitors of human leukocyte elastase and cathepsin G. Sequence variants of squash seed protease inhibitor with altered protease selectivity

    International Nuclear Information System (INIS)

    Novel peptide inhibitors of human leukocyte elastase (HLE) and cathepsin G (CG) were prepared by solid-phase peptide synthesis of P1 amino acid sequence variants of Curcurbita maxima trypsin inhibitor III (CMTI-III), a 29-residue peptide found in squash seed. A systematic study of P1 variants indicated that P1, Arg, Lys, Leu, Ala, Phe, and Met inhibit trypsin; P1, Val, Ile, Gly, Leu, Ala, Phe, and Met inhibit HLE; P1 Leu, Ala, Phe, and Met inhibit CG and chymotrypsin. Variants with P1, Val, Ile, or Gly were selective inhibitors of HLE, while inhibition of trypsin required P1 amino acids with an unbranched β carbon. Studies of Val-5-CMTI-III (P1 Val) inhibition of HLE demonstrated a 1:1 binding stoichiometry with a (Ki)app of 8.7 nM. Inhibition of HLE by Gly-5-CMTI-III indicated a significant role for reactive-site structural moieties other than the P1 side chain. Val-5-CMTI-III inhibited both HLE and human polymorphonuclear leukocyte (PMN) proteolysis of surface-bound 125I-labeled fibronectin. Val-5-CMTI-III was more effective at preventing turnover of a peptide p-nitroanilide substrate than halting dissolution of 125I-labeled fibronectin. It was about as effective as human serum α1-proteinase inhibitor in preventing PMN degradation of the connective tissue substrate. In addition to providing interesting candidates for controlling inflammatory cell proteolytic injury, the CMTI-based inhibitors are ideal for studying molecular recognition because of their small size, their ease of preparation, and the availability of sensitive and quantitative assays for intermolecular interactions

  6. Mutation analysis of the cathepsin C gene in Indian families with Papillon-Lefèvre syndrome

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    Srivastava Satish

    2003-07-01

    Full Text Available Abstract Background PLS is a rare autosomal recessive disorder characterized by early onset periodontopathia and palmar plantar keratosis. PLS is caused by mutations in the cathepsin C (CTSC gene. Dipeptidyl-peptidase I encoded by the CTSC gene removes dipeptides from the amino-terminus of protein substrates and mainly plays an immune and inflammatory role. Several mutations have been reported in this gene in patients from several ethnic groups. We report here mutation analysis of the CTSC gene in three Indian families with PLS. Methods Peripheral blood samples were obtained from individuals belonging to three Indian families with PLS for genomic DNA isolation. Exon-specific intronic primers were used to amplify DNA samples from individuals. PCR products were subsequently sequenced to detect mutations. PCR-SCCP and ASOH analyses were used to determine if mutations were present in normal control individuals. Results All patients from three families had a classic PLS phenotype, which included palmoplantar keratosis and early-onset severe periodontitis. Sequence analysis of the CTSC gene showed three novel nonsense mutations (viz., p.Q49X, p.Q69X and p.Y304X in homozygous state in affected individuals from these Indian families. Conclusions This study reported three novel nonsense mutations in three Indian families. These novel nonsense mutations are predicted to produce truncated dipeptidyl-peptidase I causing PLS phenotype in these families. A review of the literature along with three novel mutations reported here showed that the total number of mutations in the CTSC gene described to date is 41 with 17 mutations being located in exon 7.

  7. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

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    Liow, K.Y.; Chow, S.C., E-mail: chow.sek.chuen@monash.edu

    2013-11-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

  8. Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

    Science.gov (United States)

    Gikanga, Benson; Adeniji, Nia S; Patapoff, Thomas W; Chih, Hung-Wei; Yi, Li

    2016-04-20

    Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the VH, VL, or CH2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in KM or kcat values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different sites is not

  9. The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

    Science.gov (United States)

    Tsume, Yasuhiro; Amidon, Gordon L

    2012-01-01

    The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs. PMID:22450679

  10. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

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    Knopfová Lucia

    2012-03-01

    Full Text Available Abstract Background The c-Myb transcription factor is essential for the maintenance of stem-progenitor cells in bone marrow, colon epithelia, and neurogenic niches. c-Myb malfunction contributes to several types of malignancies including breast cancer. However, the function of c-Myb in the metastatic spread of breast tumors remains unexplored. In this study, we report a novel role of c-Myb in the control of specific proteases that regulate the matrix-dependent invasion of breast cancer cells. Results Ectopically expressed c-Myb enhanced migration and ability of human MDA-MB-231 and mouse 4T1 mammary cancer cells to invade Matrigel but not the collagen I matrix in vitro. c-Myb strongly increased the expression/activity of cathepsin D and matrix metalloproteinase (MMP 9 and significantly downregulated MMP1. The gene coding for cathepsin D was suggested as the c-Myb-responsive gene and downstream effector of the migration-promoting function of c-Myb. Finally, we demonstrated that c-Myb delayed the growth of mammary tumors in BALB/c mice and affected the metastatic potential of breast cancer cells in an organ-specific manner. Conclusions This study identified c-Myb as a matrix-dependent regulator of invasive behavior of breast cancer cells.

  11. The Feasibility of Enzyme Targeted Activation for Amino Acid/Dipeptide Monoester Prodrugs of Floxuridine; Cathepsin D as a Potential Targeted Enzyme

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    Gordon L. Amidon

    2012-03-01

    Full Text Available The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5¢-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0–105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5¢-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine and 5¢-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enyzmes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  12. The C-terminal subunit of artificially truncated human cathepsin B mediates its nuclear targeting and contributes to cell viability

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    Dallner Claudia

    2005-04-01

    Full Text Available Abstract Background Splicing variants of human cathepsinB primary transcripts (CB(-2,3 result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also

  13. Inhibition of cathepsin K increases modeling-based bone formation, and improves cortical dimension and strength in adult ovariectomized monkeys.

    Science.gov (United States)

    Pennypacker, Brenda L; Chen, Charles M; Zheng, Helen; Shih, Mei-Shu; Belfast, Mary; Samadfam, Rana; Duong, Le T

    2014-08-01

    Treatment with the cathepsin K (CatK) inhibitor odanacatib (ODN) protects against bone loss and maintains normal biomechanical properties in the spine and hip of ovariectomized (OVX) preclinical models. Here, we characterized the effects of ODN on the dynamics of cortical modeling and remodeling, and dimension and strength of the central femur in adult OVX-rhesus monkeys. Animals were treated with vehicle or ODN (6 or 30 mg/kg, once per day [q.d., p.o.]) in prevention mode for 21 months. Calcein and tetracycline double-labeling were given at 12 and 21 months, and the femoral cross-sections were subjected to dynamic histomorphometric and cement line analyses. ODN treatment significantly increased periosteal and endocortical bone formation (BFR/BS), accompanied with an increase in endocortical mineralizing surface (102%, p < 0.01) with the 6 mg/kg dose. ODN at both doses reduced remodeling hemiosteon numbers by 51% and 66% (p < 0.05), respectively, and ODN 30 mg/kg numerically reduced activation frequency without affecting wall thickness. On the same endocortical surface, ODN increased all modeling-based parameters, while reducing intracortical remodeling, consistent with the observed no treatment effects on cortical porosity. ODN 30 mg/kg markedly increased cortical thickness (CtTh, p < 0.001) and reduced marrow area (p < 0.01). Lastly, ODN treatment increased femoral structural strength (p < 0.001). Peak load was positively correlated with the increases in bone mineral content (BMC) (r(2)  = 0.9057, p < 0.0001) and CtTh (r2  = 0.6866, p < 0.0001). Taken together, by reducing cortical remodeling-based and stimulating modeling-based bone formation, ODN significantly improved cortical dimension and strength in OVX monkeys. This novel mechanism of CatK inhibition in stimulating cortical formation suggests that ODN represents a novel therapeutic approach for the treatment of osteoporosis. PMID:24591096

  14. Azanitrile Cathepsin K Inhibitors: Effects on Cell Toxicity, Osteoblast-Induced Mineralization and Osteoclast-Mediated Bone Resorption.

    Directory of Open Access Journals (Sweden)

    Zhong-Yuan Ren

    Full Text Available The cysteine protease cathepsin K (CatK, abundantly expressed in osteoclasts, is responsible for the degradation of bone matrix proteins, including collagen type 1. Thus, CatK is an attractive target for new anti-resorptive osteoporosis therapies, but the wider effects of CatK inhibitors on bone cells also need to be evaluated to assess their effects on bone. Therefore, we selected, among a series of synthetized isothiosemicarbazides, two molecules which are highly selective CatK inhibitors (CKIs to test their effects on osteoblasts and osteoclasts.Cell viability upon treatment of CKIs were was assayed on human osteoblast-like Saos-2, mouse monocyte cell line RAW 264.7 and mature mouse osteoclasts differentiated from bone marrow. Osteoblast-induced mineralization in Saos-2 cells and in mouse primary osteoblasts from calvaria, with or without CKIs,; were was monitored by Alizarin Red staining and alkaline phosphatase activity, while osteoclast-induced bone resorption was performed on bovine slices.Treatments with two CKIs, CKI-8 and CKI-13 in human osteoblast-like Saos-2, murine RAW 264.7 macrophages stimulated with RANKL and mouse osteoclasts differentiated from bone marrow stimulated with RANKL and MCSF were found not to be toxic at doses of up to 100 nM. As probed by Alizarin Red staining, CKI-8 did not inhibit osteoblast-induced mineralization in mouse primary osteoblasts as well as in osteoblast-like Saos-2 cells. However, CKI-13 led to a reduction in mineralization of around 40% at 10-100 nM concentrations in osteoblast-like Saos-2 cells while it did not in primary cells. After a 48-hour incubation, both CKI-8 and CKI-13 decreased bone resorption on bovine bone slices. CKI-13 was more efficient than the commercial inhibitor E-64 in inhibiting bone resorption induced by osteoclasts on bovine bone slices. Both CKI-8 and CKI-13 created smaller bone resorption pits on bovine bone slices, suggesting that the mobility of osteoclasts was slowed

  15. Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Ileana Corvo

    Full Text Available BACKGROUND: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS, we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL. Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. CONCLUSIONS/SIGNIFICANCE: These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of

  16. Cloning and expression of the cathepsin F-like cysteine protease gene in Escherichia coli and its characterization.

    Science.gov (United States)

    Joo, Han Seung; Koo, Kwang Bon; Park, Kyung In; Bae, Song Hwan; Yun, Jong Won; Chang, Chung Soon; Choi, Jang Won

    2007-04-01

    In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the 32P-labeled partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the Cys90, His226, and Asn250 residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The

  17. Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens.

    Science.gov (United States)

    Bartley, Kathryn; Huntley, John F; Wright, Harry W; Nath, Mintu; Nisbet, Alasdair J

    2012-05-01

    Vaccination is a feasible strategy for controlling the haematophagous poultry red mite Dermanyssus gallinae. A cDNA library enriched for genes upregulated after feeding was created to identify potential vaccine antigens. From this library, a gene (Dg-CatD-1) encoding a 383 amino acid protein (Dg-CatD-1) with homology to cathepsin D lysosomal aspartyl proteinases was identified as a potential vaccine candidate. A second gene (Dg-CatL-1) encoding a 341 amino acid protein (Dg-CatL-1) with homology to cathepsin L cysteine proteinases was also selected for further study. IgY obtained from naturally infested hens failed to detect Dg-CatD-1 suggesting that it is a concealed antigen. Conversely, Dg-CatL-1 was detected by IgY derived from natural-infestation, indicating that infested hens are exposed to Dg-CatL-1. Mortality rates 120 h after mites had been fed anti-Dg-CatD-1 were significantly higher than those fed control IgY (PF<0·01). In a survival analysis, fitting a proportional hazards model to the time of death of mites, anti-Dg-CatD-1 and anti-Dg-CatL-1 IgY had 4·42 and 2·13 times higher risks of dying compared with controls (PF<0·05). Dg-CatD-1 and L-1 both have potential as vaccine antigens as part of a multi-component vaccine and have the potential to be improved as vaccine antigens using alternative expression systems. PMID:22310226

  18. Upregulation of cathepsin W-expressing T cells is specific for autoimmune atrophic gastritis compared to other types of chronic gastritis

    Institute of Scientific and Technical Information of China (English)

    Doerthe Kuester; Michael Vieth; Ulrich Peitz; Stefan Kahl; Manfred Stolte; Albert Roessner; Ekkehard Weber; Peter Malfertheiner; Thomas Wex

    2005-01-01

    AIM: To investigate a pathophysiological role of cathepsin W (CatW), a putative thiol-dependent cysteine protease,which is specifically expressed in cytotoxic lymphocytes,in different types of chronic inflammation of the gastric mucosa.METHODS: Gastric and duodenal biopsies of patients with Helicobacter pylori ( H pylori)-associated active gastritis ( Hp,n = 19), chemically induced reactive gastritis (CG, n = 17),autoimmune atrophic gastritis (AIG, n = 20), lymphocytic corpus gastritis (LG, n = 29), celiac disease (CD, n = 10),and corresponding controls (n = 24) were analyzed by immunohistochemistry for the expression of CatW and CD45. Furthermore, immunohistochemical double staining with anti-CD3 and anti-cathepsin was performed for the samples of AIG.RESULTS: Median values of CatW-expressing cells among CD45-positive immune cells were between 2% and 6% for normal gastric mucosa, CG, and LG, whereas the corresponding value was significantly increased for AIG (24.7%, P<0.001) and significantly decreased for HP (0.7%, P<0.05). Double staining with anti-CD3 and antiCatW antibodies revealed that >90% of CatW-expressing cells in gastric mucosa of AIG were T cells. Duodenal mucosa had significantly more CatW/CD45-positive cells than normal gastric mucosa (median: 17.8% vs 2%, P<0.01).The corresponding proportion of CatW/CD45-positive cells was decreased in CD compared to duodenal mucosa (median: 2.1% vs 17.8%, P<0.05).CONCLUSION: The opposite findings regarding the presence of CatW-positive cells in AIG (increase) and CD (decrease) reflects the different cellular composition of immune cells involved in the pathogenesis of these diseases.

  19. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

    Directory of Open Access Journals (Sweden)

    Panat Anuracpreeda

    Full Text Available Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden.In this study, we have produced a monoclonal antibody (MoAb against recombinant F. gigantica cathepsin L1 (rFgCatL1, and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA and immunochromatographic (IC test for rapid detection of circulating cathepsin L1 protease (CatL1 in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities.The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA, and 93%, 100% and 98.2% (for IC test, while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA, and 96.7%, 100% and 99.1% (for IC test.These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

  20. Association between polymorphism in the human cathepsin L (CTSL 1) promoter with hypertension in the uygur, kazak and han population in china

    International Nuclear Information System (INIS)

    To systemically investigate the association between the polymorphism (rs3118869) in cathepsin Lenzyme gene with hypertension in three ethnic groups (Han, Kazak and Uygur) in China. Study Design: Case-control study. Place and Duration of Study: Department of Cardiology, The First Affiliated Hospital, Shihezi Medical College, Shihezi University and Department of Internal Medicine and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, from January 2013 to May 2014. Methodology: This case-control study included 1224 patients (422 Uygur, 425 Kazak and 377 Han individuals) with hypertension and 967 healthy unrelated individuals (339 Uygur, 337 Kazak and 291 Han individuals) as controls. The participants came from three ethnic groups (Han, Kazak and Uygur) which were recruited from Xinjiang Province of China. The polymorphism (rs3118869) of the human cathepsin Lgene was genotyped using the TaqMan 5' nuclease assay. Binary logistic regression was built to determine the association of polymorphism with hypertension. Results: The genotype distribution of polymorphism was not significantly different in three ethnic groups. The rs3118869 polymorphism was significantly associated with Essential Hypertension (EH) in co-dominant model (A/C vs. C/C) in total people (OR = 0.697, 95% CI = 0.520 -0.932, p = 0.015), the same result was obtained in recessive model (C/C + A/C vs. A/A) in total people (OR = 0.689, 95% CI = 0.522 -0.910, p = 0.009). Similar finding of rs3118869 in recessive model (C/C + A/C vs. A/A) was also observed after adjusting the variable to the covariates age (OR = 0.629, 95% CI = 0.464 0853, p = 0.003). Conclusion: The study results indicate the A-allele of rs3118869 is a protective factor in hypertension. (author)

  1. 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

    OpenAIRE

    Welgus, H G; Connolly, N L; Senior, R M

    1986-01-01

    Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an e...

  2. Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology.

    Science.gov (United States)

    Flanagan-Steet, Heather; Matheny, Courtney; Petrey, Aaron; Parker, Joshua; Steet, Richard

    2016-09-01

    Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and ß subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg(-/-) embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg(-/-) embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg(-/-) embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal. PMID:27241848

  3. Advances in Cathepsin K inhibitor Odanacatib in treatment of osteoporosis%组织蛋白酶K抑制剂Odanacatib治疗骨质疏松的研究进展

    Institute of Scientific and Technical Information of China (English)

    李明; 吕厚辰; 张里程; 唐佩福; 张立海

    2015-01-01

    组织蛋白酶K(cathepsin K,Cat K)为表达于破骨细胞的一种半胱氨酸蛋白酶,是骨重建过程中骨吸收的关键酶。相关研究证实,抑制其活性是治疗骨质疏松的一个新思路。当前最新研发的组织蛋白酶K抑制剂Odanacatib (ODN),可有效抑制骨的吸收且不抑制骨的形成,耐受性好,无明显的药物相关不良反应,为治疗骨质疏松提供了新的选择。%Cathepsin K (Cat K) is a cysteine protease expressed in osteoclasts, which is the key enzyme of bone resorption in the process of bone rebuilding. Related research confirms that inhibiting its activity is a new way for the treatment of osteoporosis. As the latest research and development of cathepsin K inhibitor, Odanacatib (ODN) can effectively inhibit bone absorption without inhibiting bone formation, and it also shows well tolerance and no obvious drug related adverse reaction, which provides a new option for treatment of osteoporosis.

  4. Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification.

    Science.gov (United States)

    Cheng, Tsing; Hitomi, Kiyotaka; van Vlijmen-Willems, Ivonne M J J; de Jongh, Gys J; Yamamoto, Kanae; Nishi, Koji; Watts, Colin; Reinheckel, Thomas; Schalkwijk, Joost; Zeeuwen, Patrick L J M

    2006-06-01

    Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis. PMID:16565075

  5. Fucoidan alleviates apoptosis by inhibiting cathepsin B and oxidative stress%褐藻多糖硫酸酯对组织蛋白酶B活性影响的实验研究

    Institute of Scientific and Technical Information of China (English)

    张甘霖; 朱晓新; 李萍

    2011-01-01

    目的 研究褐藻多糖硫酸酯(Fucoidan)对氧化应激损伤及溶酶体组织蛋白酶B活性的影响,探讨Fucoidan用于中枢神经系统疾病的作用机制.方法 50 μg·L-1神经生长因子(nerve growth factor,NGF)分化大鼠肾上腺嗜铬细胞瘤PC12细胞(differentiated PC12,dPC12)7 d,0.5 mmol/L H2O2刺激30 min,给予10 mg·L-1 Fucoidan干预.酶标仪检测细胞内超氧化物歧化酶(superoxide dismutase,SOD)及活性氧(reactive oxygen species,ROS)活性;荧光底物法检测细胞质中溶酶体组织蛋白酶B的活性;酶-底物法检测不同浓度Fucoidan(0.1、1、10、50、100 mg·L-1)对人肝脏溶酶体组织蛋白酶B活性的影响.结果 氧化损伤后细胞中ROS显著升高达正常水平的185.2%,伴有SOD下降,溶酶体组织蛋白酶B活性上升至正常水平的236.7%;Fucoidan对SOD活性没有明显影响,能降低ROS水平,且对细胞质内和人肝脏溶酶体组织蛋白酶B的活性均具有抑制作用.结论 Fucoidan降低活性氧含量抗氧化损伤的作用可能与其抑制溶酶体组织蛋白酶B的活性有关.%Objective To study mechanism of Fucoidan in neurodegenerative disease by observing its effect on cathepsin B activities and oxidative stress. Methods After 7 days' differentiation by 50 μg · L-1 nerve growth factor( NGF), PC12 cells were exposed to 0.5 mmol/ L H2O2 for 30 min, and then treated with Fucoidan( 10 mg · L 1). The activities of superoxide dismutase(SOD) and reactive oxygen species(ROS) inside ceils of each group were assayed. Cathepsin B activities inside cells were determined after being extracted from dPC12.Human liver cathepsin B activities were tested after being exposed to different concentrations of Fucoidan(0.1, 1, 10, 50, 100 mg · L-1 ).Results After oxidative stress, the activities of ROS inside cells increased to 185.2% of normal control, accompanied by lower SOD activities and higher activities of cathepsin B(236.7% ) in cytoplasm. Fucoidan treatment decreased ROS

  6. NMR characterization and conformational analysis of a potent papain-family cathepsin L-like cysteine protease inhibitor with different behaviour in polar and apolar media

    Science.gov (United States)

    Rotondo, Archimede; Ettari, Roberta; Zappalà, Maria; De Micheli, Carlo; Rotondo, Enrico

    2014-11-01

    We recently reported the synthesis, of a potent papain-family cathepsin L-like cysteine protease inhibitor, as new lead compound for the development of new drugs that can be used as antiprotozoal agents. The investigation of its conformational profile is crucial for the in-depth understanding of its biological behaviour. Our careful NMR analysis has been based on the complete and total assignment of 1H, 13C, 15N and 19F signals of the molecule in both CDCl3 and CD3OH, which could reproduce in some way a scenario of polar and not polar phases into the biological environment. In this way it has been unveiled a different behaviour of the molecule in polar and apolar media. In CDCl3 it is possible to define stable conformational arrangements on the basis of the detected through space contacts, whereas, in CD3OH a greater conformational freedom is envisaged: (a) by the overlap of any of the CH2 diastereotopic resonances (unable to distinguish asymmetric molecular sides because of the free rotation about the single bonded chains), (b) by the less definite measured vicinities not consistent with just one conformation and (c) by the evident loss or switching of key intramolecular hydrogen interactions.

  7. A single diamagnetic catalyCEST MRI contrast agent that detects cathepsin B enzyme activity by using a ratio of two CEST signals

    Science.gov (United States)

    Hingorani, Dina V.; Montano, Luis A.; Randtke, Edward A.; Lee, Yeon Sun; Cárdenas-Rodríguez, Julio; Pagel, Mark D.

    2016-01-01

    CatalyCEST MRI can detect enzyme activity by monitoring the change in chemical exchange with water after a contrast agent is cleaved by an enzyme. Often these molecules use paramagnetic metals and are delivered with an additional non-responsive reference molecule. To improve this approach for molecular imaging, a single diamagnetic agent with enzyme-responsive and enzyme-unresponsive CEST signals was synthesized and characterized. The CEST signal from the aryl amide disappeared after cleavage of a dipeptidyl ligand with cathepsin B, while a salicylic acid moiety was largely unresponsive to enzyme activity. The ratiometric comparison of the two CEST signals from the same agent allowed for concentration independent measurements of enzyme activity. The chemical exchange rate of the salicylic acid moiety was unchanged after enzyme catalysis, which further validated that this moiety was enzyme-unresponsive. The temperature dependence of the chemical exchange rate of the salicylic acid moiety was non-Arrhenius, suggesting a two-step chemical exchange mechanism for salicylic acid. The good detection sensitivity at low saturation power facilitates clinical translation, along with the potentially low toxicity of a non-metallic MRI contrast agent. The modular design of the agent constitutes a platform technology that expands the variety of agents that may be employed by catalyCEST MRI for molecular imaging. PMID:26633584

  8. Asparagine endopeptidase is not essential for class II MHC antigen presentation but is required for processing of cathepsin L in mice.

    Science.gov (United States)

    Maehr, René; Hang, Howard C; Mintern, Justine D; Kim, You-Me; Cuvillier, Armelle; Nishimura, Mikio; Yamada, Kenji; Shirahama-Noda, Kanae; Hara-Nishimura, Ikuko; Ploegh, Hidde L

    2005-06-01

    Class II MHC molecules survey the endocytic compartments of APCs and present antigenic peptides to CD4 T cells. In this context, lysosomal proteases are essential not only for the generation of antigenic peptides but also for proteolysis of the invariant chain to allow the maturation of class II MHC molecules. Recent studies with protease inhibitors have implicated the asparagine endopeptidase (AEP) in class II MHC-restricted Ag presentation. We now report that AEP-deficient mice show no differences in processing of the invariant chain or maturation of class II MHC products compared with wild-type mice. In the absence of AEP, presentation to primary T cells of OVA and myelin oligodendrocyte glycoprotein, two Ags that contain asparagine residues within or in proximity to the relevant epitopes was unimpaired. Cathepsin (Cat) L, a lysosomal cysteine protease essential for the development to CD4 and NK T cells, fails to be processed into its mature two-chain form in AEP-deficient cells. Despite this, the numbers of CD4 and NK T cells are normal, showing that the single-chain form of Cat L is sufficient for its function in vivo. We conclude that AEP is essential for processing of Cat L but not for class II MHC-restricted Ag presentation. PMID:15905550

  9. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Science.gov (United States)

    Gottstein, Bruno; Schneeberger, Marianne; Boubaker, Ghalia; Merkle, Bernadette; Huber, Cristina; Spiliotis, Markus; Müller, Norbert; Garate, Teresa; Doherr, Marcus G

    2014-06-01

    Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2) and cathepsin L-1 (recCL1), were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG) conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES) from adult stage liver flukes was assessed by receiver operator characteristic (ROC) analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20), patients with other parasitic infections (n=87) and patients with malignancies (n=121). The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy) employing the threshold (cut-off) to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls. PMID:24922050

  10. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Directory of Open Access Journals (Sweden)

    Bruno Gottstein

    2014-06-01

    Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

  11. A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites.

    Science.gov (United States)

    Beckham, Simone A; Piedrafita, David; Phillips, Carolyn I; Samarawickrema, Nirma; Law, Ruby H P; Smooker, Peter M; Quinsey, Noelene S; Irving, James A; Greenwood, Deanne; Verhelst, Steven H L; Bogyo, Matthew; Turk, Boris; Coetzer, Theresa H; Wijeyewickrema, Lakshmi C; Spithill, Terry W; Pike, Robert N

    2009-07-01

    The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination. PMID:19401154

  12. Complementary use of ion trap/time-of-flight mass spectrometry in combination with capillary high-pressure liquid chromatography: early characterization of in vivo metabolites of the cathepsin K inhibitor NVP-AAV490 in rat.

    Science.gov (United States)

    Blum, Wolfgang; Buhl, Thomas; Altmann, Eva; Kühnöl, Jürgen; Ramstein, Philippe; Aichholz, Reiner

    2003-04-25

    Cathepsin K is a cysteine proteinase, primarily expressed in osteoclasts, which has a strong collagenolytic activity and plays an essential role involved in bone matrix degradation. Its inhibition could provide a novel approach to the treatment and prevention of osteoporosis. One structural class of lead compounds in our cathepsin K inhibitors program is based on an arylaminoethyl amide scaffold, which has potential metabolic weak points that might be stabilized by appropriate chemical modification(s). For the identification of potential metabolic "soft spots" and the rational design of improved derivatives, early biotransformation of a potent arylaminoethyl amide cathepsin K inhibitor (NVP-AAV490-NX) was investigated in plasma, urine and liver homogenates of rats after intravenous bolus administration of 10 mg/kg. The detection and identification of metabolites was achieved by high-resolution mass spectrometry (time-of-flight MS) and multi-dimensional mass spectrometry (ion trap MS). Both mass spectrometers were combined with reversed-phase capillary high-performance liquid chromatography columns. It was demonstrated that both mass analyzers complement each other and that, even in the sub-nanogram range, the resulting set of MS data can be successfully used to elucidate most of the metabolic changes unambiguously, solely by mass spectrometric techniques. The proposed metabolite structures were additionally corroborated by exact mass measurement of the protonated molecular ions to confirm the predicted elemental composition, by determination of the number of the exchangeable hydrogen atoms replacing water against deuterium oxide as mobile phase and, in one case, by an MS(3) product ion experiment in order to elucidate the site of conjugation. PMID:12650749

  13. Going Beyond Common Drug Metabolizing Enzymes: Case Studies of Biotransformation Involving Aldehyde Oxidase, γ-Glutamyl Transpeptidase, Cathepsin B, Flavin-Containing Monooxygenase, and ADP-Ribosyltransferase.

    Science.gov (United States)

    Fan, Peter W; Zhang, Donglu; Halladay, Jason S; Driscoll, James P; Khojasteh, S Cyrus

    2016-08-01

    The significant roles that cytochrome P450 (P450) and UDP-glucuronosyl transferase (UGT) enzymes play in drug discovery cannot be ignored, and these enzyme systems are commonly examined during drug optimization using liver microsomes or hepatocytes. At the same time, other drug-metabolizing enzymes have a role in the metabolism of drugs and can lead to challenges in drug optimization that could be mitigated if the contributions of these enzymes were better understood. We present examples (mostly from Genentech) of five different non-P450 and non-UGT enzymes that contribute to the metabolic clearance or bioactivation of drugs and drug candidates. Aldehyde oxidase mediates a unique amide hydrolysis of GDC-0834 (N-[3-[6-[4-[(2R)-1,4-dimethyl-3-oxopiperazin-2-yl]anilino]-4-methyl-5-oxopyrazin-2-yl]-2-methylphenyl]-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide), leading to high clearance of the drug. Likewise, the rodent-specific ribose conjugation by ADP-ribosyltransferase leads to high clearance of an interleukin-2-inducible T-cell kinase inhibitor. Metabolic reactions by flavin-containing monooxygenases (FMO) are easily mistaken for P450-mediated metabolism such as oxidative defluorination of 4-fluoro-N-methylaniline by FMO. Gamma-glutamyl transpeptidase is involved in the initial hydrolysis of glutathione metabolites, leading to formation of proximate toxins and nephrotoxicity, as is observed with cisplatin in the clinic, or renal toxicity, as is observed with efavirenz in rodents. Finally, cathepsin B is a lysosomal enzyme that is highly expressed in human tumors and has been targeted to release potent cytotoxins, as in the case of brentuximab vedotin. These examples of non-P450- and non-UGT-mediated metabolism show that a more complete understanding of drug metabolizing enzymes allows for better insight into the fate of drugs and improved design strategies of molecules in drug discovery. PMID:27117704

  14. Towards delineating functions within the fasciola secreted cathepsin l protease family by integrating in vivo based sub-proteomics and phylogenetics.

    Directory of Open Access Journals (Sweden)

    Russell M Morphew

    Full Text Available BACKGROUND: fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES products are also the most promising vaccine candidates, the cathepsin L (Cat L protease family. METHODOLOGY/PRINCIPAL FINDINGS: the sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. CONCLUSIONS/SIGNIFICANCE: we have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.

  15. Lipopolysaccharide-Mediated Induction of Concurrent IL-1β and IL-23 Expression in THP-1 Cells Exhibits Differential Requirements for Caspase-1 and Cathepsin B Activity.

    Science.gov (United States)

    Wynick, Christopher; Petes, Carlene; Tigert, Alexander; Gee, Katrina

    2016-08-01

    The inflammasome is a multimeric protein complex required for interleukin (IL)-1β production. Upon lipopolysaccharide (LPS) triggering of toll-like receptor (TLR)-4 and subsequent ATP signaling, the NOD-like receptor containing-pyrin domain 3 (NLRP3) inflammasome is activated to cleave pro-caspase-1 into caspase-1, allowing the secretion of IL-1β. IL-1β is known to function with IL-23 in the regulation of IL-17-producing CD4(+) T cells, Th17 cells, in adaptive immunity. Recently, studies have shown that IL-1β and IL-23 together activate IL-17-producing innate lymphoid cells, demonstrating that the pair may exhibit additional effects on cell differentiation. Using an in vitro model of bacterial infection, LPS treatment of human monocytic cells, we investigated the molecular mechanisms involved in the co-expression of IL-1β and IL-23. We found that IL-1β is partially required for optimal LPS-induced IL-23 production. We also found that IL-23 production was partially dependent on ATP signaling via the P2X7 receptor, whereas IL-1β production required this signaling. Furthermore, we identified a novel role for cathepsin B activity in IL-23 production. Taken together, this study identifies differential requirements for the co-expression of IL-1β and IL-23. Due to their similar roles in Th17 differentiation, characterization of the regulatory mechanisms for LPS-induced IL-1β and IL-23 may reveal novel information into the pathology of the inflammatory response particularly during bacterial infection. PMID:27096899

  16. Peroxisome Proliferator-activated Receptor γ Induces Apoptosis and Inhibits Autophagy of Human Monocyte-derived Macrophages via Induction of Cathepsin L

    Science.gov (United States)

    Mahmood, Dler Faieeq Darweesh; Jguirim-Souissi, Imene; Khadija, El-Hadri; Blondeau, Nicolas; Diderot, Vimala; Amrani, Souliman; Slimane, Mohamed-Naceur; Syrovets, Tatiana; Simmet, Thomas; Rouis, Mustapha

    2011-01-01

    Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) γ, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPARγ might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPARγ activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPARγ by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPARγ-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPARγ-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPARγ can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis. PMID:21700710

  17. Optical Imaging with a Cathepsin B Activated Probe for the Enhanced Detection of Esophageal Adenocarcinoma by Dual Channel Fluorescent Upper GI Endoscopy

    Directory of Open Access Journals (Sweden)

    Peiman Habibollahi, Jose-Luiz Figueiredo, Pedram Heidari, Austin M Dulak, Yu Imamura, Adam J. Bass, Shuji Ogino, Andrew T Chan, Umar Mahmood

    2012-01-01

    Full Text Available Despite significant advances in diagnosis and treatment, the prognosis of esophageal adenocarcinoma remains poor highlighting the importance of early detection. Although white light (WL upper endoscopy can be used for screening of the esophagus, it has limited sensitivity for early stage disease. Thus, development of new imaging technology to improve the diagnostic capabilities of upper GI endoscopy for early detection of esophageal adenocarcinoma is an important unmet need. The goal of this study was to develop a method for the detection of malignant lesions in the esophagus using WL upper endoscopy combined with near infrared (NIR imaging with a protease activatable probe (Prosense750 selective for cathepsin B (CTSB. An orthotopic murine model for distal esophageal adenocarcinoma was generated through the implantation of OE-33 and OE-19 human esophageal adenocarcinoma lines in immunocompromised mice. The mice were imaged simultaneously for WL and NIR signal using a custom-built dual channel upper GI endoscope. The presence of tumor was confirmed by histology and target to background ratios (TBR were compared for both WL and NIR imaging. NIR imaging with ProSense750 significantly improved upon the TBRs of esophageal tumor foci, with a TBR of 3.64±0.14 and 4.50±0.11 for the OE-33 and OE-19 tumors respectively, compared to 0.88±0.04 and 0.81±0.02 TBR for WL imaging. The combination of protease probes with novel imaging devices has the potential to improve esophageal tumor detection by fluorescently highlighting neoplastic regions.

  18. Mycobacterium bovis BCG Interferes with miR-3619-5p Control of Cathepsin S in the Process of Autophagy

    Science.gov (United States)

    Pawar, Kamlesh; Sharbati, Jutta; Einspanier, Ralf; Sharbati, Soroush

    2016-01-01

    Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637, and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages. PMID:27014637

  19. Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

    Science.gov (United States)

    Zhou, Nan; Pan, Ting; Zhang, Junsong; Li, Qianwen; Zhang, Xue; Bai, Chuan; Huang, Feng; Peng, Tao; Zhang, Jianhua; Liu, Chao; Tao, Liang; Zhang, Hui

    2016-04-22

    Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. PMID:26953343

  20. The Prognostic Significance of a Combined Determination of Cathepsin D and Estrogen Receptors in Breast Carcinomas with Positive Axillary Lymph Nodes

    Institute of Scientific and Technical Information of China (English)

    Yun Niu; Xue Yang; Yu Fan; Ajuan Lü; Tieju Liu; Xilin Fu

    2006-01-01

    OBJECTIVE The aim of this study was to investigate the correlation between cathepsin D (Cath-D) and estrogen receptor (ER)expression in breast cancer tissue and to explore the prognostic significance of their combined determination in breast carcinoma patients with positive axillary lymph nodes. METHODS One hundred and thirty-eight cases of breast carcinoma were examined by immunohistochemistry (IHC) and the results relating to patient follow-up analyzed.RESULTS The overall 5-year disease-free survival rate (DFS) was 60.9% (84/138) in the series. The positive rate of Cath-D expression in the tumor cells was 55.07% and the positive ER staining was 51.4%. A definite significant negative correlation was found between the positive rates for Cath-D and ER (r=-0.294, P=0.001) The Cath-D expression for the cases in clinical Stage Ⅱ, ≥10 positive-node and recurrence or distant metastasis, was higher than that those cases in clinical Stage Ⅱ with fewer node-metastasis and with 5 year DFS (χ2=13.926, P=0.000;χ2=13.070, P=0.001; χ2=10.545, P=0.001). However, there was no significant difference of Cath-D expression between 2 groups of patients with different ages or among the different histopathologic types of the nonspecific invasive carcinoma. In the combined examination of Cath-D and ER, the cases that were ER (+) and Cath-D (-) had the highest 5-year DFS compared to other situations. In contrast, the cases that were reversed in expression, ie, ER(-) and Cath-D(+), had a lower 5-year DFS.There was a significant difference between the 2 conditions (χ2=18.675,P=0.000).CONCLUSION A combined determination and analysis of Cath-D and ER expression may be more useful to establish a prognosis than the biological characteristics of carcinomas with positive lymph nodes.

  1. Effects of 16-month treatment with the cathepsin K inhibitor ONO-5334 on bone markers, mineral density, strength and histomorphometry in ovariectomized cynomolgus monkeys.

    Science.gov (United States)

    Yamada, Hiroyuki; Ochi, Yasuo; Mori, Hiroshi; Nishikawa, Satoshi; Hashimoto, Yasuaki; Nakanishi, Yasutomo; Tanaka, Makoto; Bruce, Mark; Deacon, Steve; Kawabata, Kazuhito

    2016-05-01

    We examined the effects of ONO-5334, a cathepsin K inhibitor, on bone markers, BMD, strength and histomorphometry in ovariectomized (OVX) cynomolgus monkeys. ONO-5334 (1.2, 6 and 30mg/kg/day, p.o.), alendronate (0.05mg/kg/2weeks, i.v.), or vehicle was administered to OVX monkeys (all groups N=20) for 16months. A concurrent Sham group (N=20) was also treated with vehicle for 16months. OVX significantly increased bone resorption and formation markers and decreased BMD in lumbar vertebra, femoral neck, proximal tibia and distal radius. Alendronate suppressed these parameters to a level similar to that in the Sham-operated monkeys. ONO-5334 at doses 6 and 30mg/kg decreased bone resorption markers to a level roughly half of that in the Sham group, while keeping bone formation markers level above that in the Sham monkeys. Changes in DXA BMD confirmed that ONO-5334 at doses 6 and 30mg/kg increased BMD to a level greater than that in the Sham group in all examined sites. In the proximal tibia, in vivo pQCT analysis showed that ONO-5334 at doses 6 and 30mg/kg suppressed trabecular BMD loss to the sham level. However, ONO-5334 increased cortical BMD, cortical area and cortical thickness to a level greater than that in the Sham group, suggesting that ONO-5334 improves both cortical BMD and cortical geometry. Histomorphometric analysis revealed that ONO-5334 suppressed bone formation rate (BFR) at osteonal site in the midshaft femur but did not influence OVX-induced increase in BFR at either the periosteal or endocortical surfaces. Unlike alendronate, ONO-5334 increased osteoclasts surface (Oc.S/BS) and serum tartrate-resistant acid phosphatise 5b (TRAP5b) activity, highlighting the difference in the mode of action between these two drugs. Our results suggest that ONO-5334 has therapeutic potential not only in vertebral bones, but also in non-vertebral bones. PMID:26921823

  2. An oral cathepsin K inhibitor ONO-5334 inhibits N-terminal and C-terminal collagen crosslinks in serum and urine at similar plasma concentrations in postmenopausal women.

    Science.gov (United States)

    Tanaka, Makoto; Hashimoto, Yoshitaka; Hasegawa, Chihiro

    2015-12-01

    Relationships between the plasma concentration of a cathepsin K inhibitor (ONO-5334) and inhibition of bone resorption markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in serum and urinary NTX/creatinine and CTX/creatinine were examined in 10 postmenopausal women. The subjects received slow-release tablets of 100mg ONO-5534 under fasted or fed conditions in a study with a crossover design. Inhibition of serum NTX and CTX levels and plasma concentrations of ONO-5334 were monitored at 0, 24, 48 and 168 h after dosing. Changes in urinary NTX/creatinine and CTX/creatinine levels in second morning urine were evaluated on 0, 1, 2 and 7 days after dosing. Data were analyzed using sigmoid maximal drug effect (Emax) models. The maximal inhibition, estimated Emax values, were -31.8% for serum NTX, -53.1% for serum CTX, -67.2% for urinary NTX/creatinine, and -95.2% for urinary CTX/creatinine. The estimated half maximal effective plasma concentrations (EC50) of ONO-5334 and confidence intervals were 1.79 (1.01 to 3.16) ng/mL for serum NTX, 2.07 (1.63 to 2.62) ng/mL for serum CTX, 1.85 (1.30 to 2.61) ng/mL for urinary NTX/creatinine, and 1.98 (0.94 to 3.76) ng/mL for urinary CTX/creatinine. EC50 values for the four crosslinks did not significantly differ, as indicated by the overlapping 95% confidence intervals. The highest signal-to-noise ratio was achieved with serum CTX, and was 2-fold higher than that on serum NTX. Inhibition for serum NTX and CTX, and urinary NTX/creatinine and CTX/creatinine by ONO-5334 were all correlated with correlation coefficients ranging from 0.55 to 0.80. In conclusion, data of ONO-5334 slow-releasing tablets in postmenopausal women were well fitted in Emax model. In all measured telopeptides, the maximal inhibition was obtained at urinary CTX/creatinine level, but serum CTX had the highest signal-to-noise ratio. Inhibition for all measured telopeptides by ONO-5334 were all correlated. The estimated half

  3. 海湾扇贝组织蛋白酶L基因编码区的克隆和分析%Cloning and sequence analysis of cathepsin L gene from bay scallop Argopecten irradians

    Institute of Scientific and Technical Information of China (English)

    李娟; 李莉; 张国范

    2011-01-01

    通过cDNA末端快速扩增技术(RACE),从海湾扇贝(Argopecten irradians)中克隆得到了组织蛋白酶L基因(AiCL)的编码区全长,为1095 bp,推测编码364个氨基酸.经比对与分析发现蛋白序列中存在4个组织蛋白酶L活性位点保守氨基酸:Q164,C170,H309,N329;6个极为保守的半胱氨酸残基:C167,C201,C210,C243,C302,C351.预测其N端17个氨基酸为信号肽序列,C端ASYPTV可能也是分泌信号.AiCL成熟蛋白分子由219个氨基酸构成,前体肽的切割位点预计在A145和M146之间.序列同源性分析中,AiCL蛋白序列与软体动物同源蛋白最为相似,序列一致性在50%以上,在系统进化树中与其它无脊椎动物组织蛋白酶L聚合到一起.通过SwISS-MODEL构建的AiCL成熟蛋白三维模型表明该蛋白空间结构高度保守.根据其序列特征,推测~CL可能具有水解多种肌蛋白的活性.%The complete open reading frame (ORF) of cathepsin L was obtained from bay scallop (Argopecten irradians) by RACE technique. The ORF was of 1 095 bp, encoding 364 amino acids. Four conserved residues for enzyme activity (Q164, C170, H309, N329) and six preserved cysteines were found in the protein sequence. 17 amino acids of N terminal were predicted to be the signal peptide and the sequence ASYPTV at C terminal was found to be a possible signal sequence. The mature protein of AICL contained 219 amino acids and the potential cleavage site was between A 145 and M 146. Analysis indicated AiCL shared the highest identities (>50%) with molluscan cathepsin Ls.It clustered with other invertebrate cathepsin Ls in the phylogenetic tree. The 3D-model predicted by SWISS-MODEL demonstrated that cathepsin L was conserved in tertiary structure. Collectively, data showed that AiCL was likely to catalyze the hydrolysis of multiple muscle related proteins.

  4. 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

    Science.gov (United States)

    Welgus, H G; Connolly, N L; Senior, R M

    1986-05-01

    Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is

  5. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K; Constituintes quimicos das cascas do caule de Vochysia thyrsoidea Pohl. (Vochysiaceae) e avaliacao das atividades citotoxica e inibitoria frente as catepsinas B e K

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, Lorena Ramos Freitas de; Silva, Jame' s A. da; Vieira, Paulo Cezar [Universidade Federal de Sao Carlos (UFSCar), SP (Brazil). Departamento de Quimica; Costa, Maisa Borges; Santos, Mirley Luciene dos; Menezes, Antonio Carlos Severo, E-mail: amenezes@ueg.br [Universidade Estadual de Goias (UEG), Anapolis, GO (Brazil). Unidade Universitaria de Ciencias Exatas e Tecnologicas; Sbardelotto, Aline Borba; Pessoa, Claudia do O; Moraes, Manoel Odorico de [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil). Fac. de Medicina. Dept. de Fisiologia e Farmacologia

    2014-04-15

    A new flavonoid, catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC{sub 50} = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC{sub 50} = 36.80 μM and IC{sub 50} = 25.37 μM, respectively (author)

  6. The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

    Science.gov (United States)

    Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

    2015-01-01

    In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

  7. Treatment with Recombinant Trichinella spiralis Cathepsin B-like Protein Ameliorates Intestinal Ischemia/Reperfusion Injury in Mice by Promoting a Switch from M1 to M2 Macrophages.

    Science.gov (United States)

    Liu, Wei-Feng; Wen, Shi-Hong; Zhan, Jian-Hua; Li, Yun-Sheng; Shen, Jian-Tong; Yang, Wen-Jing; Zhou, Xing-Wang; Liu, Ke-Xuan

    2015-07-01

    Intestinal ischemia/reperfusion (I/R) injury, in which macrophages play a key role, can cause high morbidity and mortality. The switch from classically (M1) to alternatively (M2) activated macrophages, which is dependent on the activation of STAT6 signaling, has been shown to protect organs from I/R injuries. In the current study, the effects of recombinant Trichinella spiralis cathepsin B-like protein (rTsCPB) on intestinal I/R injury and the potential mechanism related to macrophage phenotypes switch were investigated. In a mouse I/R model undergoing 60-min intestinal ischemia followed by 2-h or 7-d reperfusion, we demonstrated that intestinal I/R caused significant intestinal injury and induced a switch from M2 to M1 macrophages, evidenced by a decrease in levels of M2 markers (arginase-1 and found in inflammatory zone protein), an increase in levels of M1 markers (inducible NO synthase and CCR7), and a decrease in the ratio of M2/M1 macrophages. RTsCPB reversed intestinal I/R-induced M2-M1 transition and promoted M1-M2 phenotype switch evidenced by a significant decrease in M1 markers, an increase in M2 markers, and the ratio of M2/M1 macrophages. Meanwhile, rTsCPB significantly ameliorated intestinal injury and improved intestinal function and survival rate of animals, accompanied by a decrease in neutrophil infiltration and an increase in cell proliferation in the intestine. However, a selective STAT6 inhibitor, AS1517499, reversed the protective effects of rTsCPB by inhibiting M1 to M2 transition. These findings suggest that intestinal I/R injury causes a switch from M2 to M1 macrophages and that rTsCPB ameliorates intestinal injury by promoting STAT6-dependent M1 to M2 transition. PMID:25987744

  8. Clone, expression, purification and activity characterization of Jian carp(Cyprinus carpio var.Jian) Cathepsin L%建鲤组织蛋白酶L的克隆表达、纯化及活性特征鉴定

    Institute of Scientific and Technical Information of China (English)

    李冉; 陈治光; 蒋然然; 陈秀华; 李树红; 李新; 钟海霞; 但静

    2015-01-01

    首先采用TA克隆技术克隆建鲤组织蛋白酶L(Cathepsin L,CAT L)成熟肽基因片段并进行双酶切鉴定,进而构建表达载体CAT L-pET-30a并转入宿主菌E.coli BL21,经1 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在37℃诱导2h表达重组CAT L蛋白.而后经尿素梯度洗涤和镍离子亲和层析纯化目的蛋白,并利用SDS-PAGE检测诱导效果和纯化过程.最后以荧光合成肽底物(Z-Phe-Arg-MCA)测活法鉴定建鲤重组CAT L的热稳定性、pH稳定性,以及鱼糜生产和冻藏中常用添加剂对其活性稳定性的影响.双酶切鉴定结果表明成功克隆了目的基因片段,与鲤鱼CAT L基因序列相似性为99.11%.SDS-PAGE分析表明经诱导、尿素梯度洗涤及亲和层析后,成功获得高度纯化目的蛋白,分子量约28 ku.活性鉴定结果表明重组CAT L在20~50℃及pH3.0~6.5范围内稳定;氯化钠、焦磷酸钠对重组CAT L活性的抑制作用呈现剂量依赖关系,而各浓度蔗糖、山梨醇则对其活性无明显作用.本研究成功克隆、表达和纯化了建鲤CAT L,并阐明了热、pH及鱼糜生产和冻藏中常用添加剂对该酶稳定性的不同影响.

  9. Análisis in silico de la catepsina B de Fasciola hepatica como diana terapéutica (In silico analysis of cathepsin B Fasciola hepatica as a therapeutic target

    Directory of Open Access Journals (Sweden)

    Naranjo-Feliciano, Dany

    2009-02-01

    Full Text Available ResumenLa fascioliasis es una enfermedad de importancia médico veterinaria,considerada por varios autores como una parasitosis emergente. Laproteína catepsina B es un producto de excreción-secreción presente en los estadios juveniles del parásito Fasciola hepatica, blanco potencial en la terapia de la fascioliasis. La forma exponencial de acumulación de secuencias de genes y proteínas de varios helmintos requiere nuevos métodos de análisis de estos datos que permitan la identificación de moléculas capaces de inducir protección inmunológica o ser utilizadas como dianas terapéuticas. Dado el desarrollo de herramientas bioinformáticas, capacidades de cómputo y métodos computacionales para la predicción de estructuras de proteínas, es posible suplir la información estructural, aun no disponible en el PDB (del inglés Protein Data Bank,con el empleo de programas de modelación por comparación. Los modelos calculados por comparación y las simulaciones de dinámica molecular pueden servir como materiales para el desarrollo de ensayos computacionales y brindar un soporte teórico para las investigaciones experimentales.SummaryThe infection caused by the trematode Fasciola hepatica is a disease of veterinary medical importance, considered by several authors as anemergent parasitism. Cathepsin B protein is an excretion-secretionproduct present in the youthful stages of the parasite, a potential target in the therapy of fascioliasis. The exponential accumulation of gene and protein sequences of several worms requires new methods of analysis of these data to allow the identification of molecules capable of inducing immune protection or be used as therapeutic targets. Given the development of bioinformatics tools, computer skills and computational methods for predicting protein structures, it is possible to substitute the structural information, not yet available in the PDB (Protein Data Bank, with the use of modeling programs

  10. 组织蛋白酶B在膀胱移行细胞癌中的表达及意义%Expression of Cathepsin B in Bladder Transitional Cell Carcinoma Tissues and Its Significance

    Institute of Scientific and Technical Information of China (English)

    石涛; 高艳; 郭永连

    2012-01-01

    Objective To investigate the expression of cathepsin B (CB) in the bladder transitional cell carcinoma (TCC) tissues and the relation between it with the invasion of TCC. Methods Forty samples of TCC including 23 of grade I and 17 grade Ⅱto Ⅲ were studied. Among them, 25 were superficial type (Tis Ta, T1), and 15 were invasion type (T2-4). Another 10 normal samples were designated as the control group. Immunohistochemical staining of CB in TCC and normal tissues was carried out by the streptavidin-biotin method. Results There were no apparent coloring in the normal bladder tissue ground substance. In TCC tissues, CB could dye the cells and a portion of ground substance. CB was positive in part of the capillary endothelial cells and fibroblasts, and in peritumoral vascular endothelial cells, the positive expression of CB was enhanced. Positive diffusion staining existed in high grade and high stage cancers. The proportions of CB staining cells for grade Ⅰ cancer tissues, gradeⅡ -Ⅲ cancer tissues, TCC superficial type of tissues, TCC invasion type of tissues and normal tissues were respectively 10.53% ± 3.76%, 21.52% ± 3.58%, 11.32% ± 2.69%, 20.57% ± 3.25%, and 0.11% ± 0.18%. The mean value of cells of CB staining was significantly higher in cancer tissues than in normal tissues (P < 0.01). The mean value of positive CB staining in grade II - IE cancer tissues was significantly higher than that in grade I cancer tissues (P < 0.01). The mean value of posive CB staining in stage T2-4 cancer tissues was significantly higher than that in superficial stage Tls-1 cancer tissues (P < 0.01). Conclusion CB should become a important marker to determine TCC development and its prognosis.%目的 探讨组织蛋白酶B(CB)在膀胱移行细胞癌(TCC)中的表达以及其与TCC浸润的关系.方法 取TCC标本40例,TCC分级Ⅰ级23例,Ⅱ~Ⅲ级17例;表浅型TCC(Tts,Ta,T1期)25例,浸润型TCC(T2~4期)15例.另取10例正常膀胱组织作为对照.用

  11. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in...

  12. Cathepsin B as a potential prognostic and therapeutic marker for human lung squamous cell carcinoma

    OpenAIRE

    Gong, Fengming; PENG, XINGCHEN; Luo, Can; Shen, Guobo; Zhao, Chengjian; ZOU, LIQUN; Li, Longhao; Sang, Yaxiong; Zhao, Yuwei; Zhao, Xia

    2013-01-01

    Background The lung squamous cell carcinoma survival rate is very poor despite multimodal treatment. It is urgent to discover novel candidate biomarkers for prognostic assessment and therapeutic targets to lung squamous cell carcinoma (SCC). Results Herein a two-dimensional gel electrophoresis and ESI-Q-TOF MS/MS-based proteomic approach was used to identify differentially expressed proteins between lung SCC and adjacent normal tissues. 31 proteins with significant alteration were identified....

  13. Inhibiting Periapical Lesions through AAV-RNAi Silencing of Cathepsin K

    OpenAIRE

    Gao, B.; Chen, W.; Hao, L; Zhu, G.; Feng, S.; Ci, H.; Zhou, X.; Stashenko, P.; Li, Y. P.

    2013-01-01

    Dental caries, one of the most prevalent infectious diseases worldwide, affects approximately 80% of children and the majority of adults. Dental caries may result in endodontic disease, leading to dental pulp necrosis, periapical inflammation and bone resorption, severe pain, and tooth loss. Periapical inflammation may also increase inflammation in other parts of the body. Although many studies have attempted to develop therapies for this disease, there is still an urgent need for effective t...

  14. Vaccine potential of recombinant cathepsinL1G against Fasciola gigantica in mice.

    Science.gov (United States)

    Changklungmoa, Narin; Phoinok, Natthacha; Yencham, Chonthicha; Sobhon, Prasert; Kueakhai, Pornanan

    2016-08-15

    In this study, we characterized and investigated the vaccine potential of FgCatL1G against Fasciola gigantica infection in mice. Recombinant mature FgCatL1G (rmFgCatL1G) was expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rmFgCatL1G combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The percents of protection of rmFgCatL1G vaccine were estimated to be 56.5% and 58.3% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immunoblot to react with the native FgCatL1s in the extract of all stages of parasites and rmFgCatL1H, recombinant pro - FgCatL1 (rpFgCatL1). By immunohistochemistry, the immune sera also reacted with FgCatL1s in the caecal epithelial cells of the parasites. The levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, were also increased with IgG1 predominating. The levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) in rmFgCatL1G-immunized group showed no significant difference from the control groups, but pathological lesions of livers in rmFgCatL1G-immunized group showed significant decrease when compared to the control groups. This study indicates that rmFgCatL1G has a vaccine potential against F. gigantica in mice, and this potential will be tested in larger livestock animals. PMID:27514897

  15. Macrophage derived cystatin B/cathepsin B in HIV replication and neuropathogenesis

    OpenAIRE

    Rivera, Linda E; Colón, Krystal; Cantres-Rosario, Yisel M; Zenón, Frances M; Meléndez, Loyda M

    2014-01-01

    Mononuclear phagocytes including monocytes and macrophages, are important defense components of innate immunity, but can be detrimental in HIV-1 infection by serving as the principal reservoirs of virus in brain and triggering a strong immune response. These viral reservoirs represent a challenge to HIV-1 eradication since they continue producing virus in tissue despite antiretroviral therapy. HIV-1 associated neurocognitive disorders (HAND) involves alterations to the blood-brain barrier and...

  16. Adsorption of Cathepsin B-sensitive peptide conjugated DOX on nanodiamonds

    International Nuclear Information System (INIS)

    Drug delivery mediated by nanodiamonds (NDs) has shown great promise in controlled drug release field. In present study, dipeptide (Phe-Lys) conjugated antitumor drug doxorubicin hydrochloride (DOX) with self-immolative p-aminobenzylcarbonyl (PABC) spacer was non-covalently bound to carboxylated NDs via the electrostatic interactions. HIV-1 trans-activating transcriptor peptide (TAT) was additionally integrated to this ND-based delivery system in order to enhance the transmembrane efficiency. Fourier transforms infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and zeta potentials were applied to characterize the DOX and TAT loaded ND delivery platform. The adsorption equilibrium, kinetics and thermodynamics for the adsorption of peptide conjugated DOX onto NDs were investigated. It was found that the adsorption fitted well with the Freundlich model and conformed to pseudo-second order kinetics. It also showed that the adsorption was a spontaneous and exothermic process. Therefore, our work offered a facile way to formulate a ND-based drug delivery platform with multifunctionality in a layer by layer adsorption fashion.

  17. Autophagy/Xenophagy as a survival strategy of cancer cells. The role of Cathepsins

    International Nuclear Information System (INIS)

    Macroautophagy, often referred as to autophagy (self-cannibalism), designates the genetically determined process by which portions of the cytoplasm, organelles and long-lived proteins are engulfed in double-membraned vacuoles (autophagosomes) and sent for lysosomal degradation. Basal levels of autophagy contribute to the maintenance of intracellular homoeostasis by ensuring the turnover of supernumerary, aged and/or damaged components. Under conditions of starvation, the autophagic pathway operates to supply cells with metabolic substrates, and thus represents an important pro-survival mechanism. In cultured cells, the withdrawal of growth factors, known to represent an experimental condition triggering autophagy, can also enhance xeno-cannibalism (xenophagy; xeno is from ancient greek=foreign)

  18. Characterization of Gut-associated Cathepsin D Hemoglobinase from Tick Ixodes ricinus (IrCD1)

    Czech Academy of Sciences Publication Activity Database

    Sojka, Daniel; Franta, Zdeněk; Frantová, Helena; Bartošová, Pavla; Horn, Martin; Váchová, Jana; O’Donoghue, A. J.; Eroy-Reveles, A. A.; Craik, C. S.; Knudsen, G. M.; Caffrey, C. R.; McKerrow, J. H.; Mareš, Michael; Kopáček, Petr

    2012-01-01

    Roč. 287, č. 25 (2012), s. 21152-21163. ISSN 0021-9258 R&D Projects: GA AV ČR IAA600960910; GA MŠk(CZ) LC06009; GA AV ČR KJB600960911; GA ČR GPP502/11/P682; GA AV ČR IAA400550705; GA ČR(CZ) GAP207/10/2183 Institutional research plan: CEZ:AV0Z60220518 Institutional support: RVO:61388963 Keywords : RHIPICEPHALUS BOOPHILUS MICROPLUS * HARD TICK * ORNITHODOROS-MOUBATA * ASPARTIC PROTEINASE * RECOGNITION DOMAIN * MOLECULAR- CLONING * ACTIVE-SITE * EXPRESSION * INHIBITOR * PROTEASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.651, year: 2012

  19. SwissProt search result: AK070448 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070448 J023048F12 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 2e-33 ...

  20. SwissProt search result: AK072767 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072767 J023138P12 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 3e-45 ...

  1. SwissProt search result: AK098910 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK098910 J013002H09 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 3e-55 ...

  2. SwissProt search result: AK104296 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK104296 001-008-H03 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 4e-60 ...

  3. SwissProt search result: AK071948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071948 J013078J21 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-37 ...

  4. SwissProt search result: AK099269 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099269 J023020I09 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 1e-50 ...

  5. SwissProt search result: AK111210 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK111210 002-179-A06 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 4e-24 ...

  6. SwissProt search result: AK099197 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099197 J023120K15 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 5e-44 ...

  7. SwissProt search result: AK119182 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119182 001-037-H03 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-47 ...

  8. SwissProt search result: AK110832 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110832 002-172-A05 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 2e-28 ...

  9. SwissProt search result: AK243212 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243212 J100042M20 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-25 ...

  10. SwissProt search result: AK099358 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099358 J033044I21 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-55 ...

  11. SwissProt search result: AK241017 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK241017 J065054E09 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 4e-20 ...

  12. SwissProt search result: AK110832 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110832 002-172-A05 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 9e-30 ...

  13. SwissProt search result: AK109160 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109160 002-155-G07 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 7e-58 ...

  14. UniProt search blastx result: AK289220 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK289220 J100065H20 P49935|CATH_MOUSE Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] - Mus musculus (Mouse) 3.00E-39 ...

  15. SwissProt search result: AK099269 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099269 J023020I09 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-46 ...

  16. SwissProt search result: AK110974 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110974 002-174-C02 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 2e-31 ...

  17. SwissProt search result: AK062382 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062382 001-102-B10 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 7e-43 ...

  18. SwissProt search result: AK066748 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066748 J013074D19 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 3e-87 ...

  19. SwissProt search result: AK100225 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100225 J023047M19 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 3e-18 ...

  20. SwissProt search result: AK072235 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK072235 J023001J24 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 2e-47 ...

  1. SwissProt search result: AK098908 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK098908 J013002F17 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 1e-46 ...

  2. SwissProt search result: AK108469 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK108469 002-143-E04 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 7e-21 ...

  3. SwissProt search result: AK061319 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061319 006-302-E09 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 2e-22 ...

  4. SwissProt search result: AK105612 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK105612 001-200-B06 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 4e-46 ...

  5. SwissProt search result: AK064820 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK064820 J013000E23 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 3e-18 ...

  6. SwissProt search result: AK071948 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071948 J013078J21 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 6e-39 ...

  7. SwissProt search result: AK062382 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK062382 001-102-B10 (P49935) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_MOUSE 7e-41 ...

  8. SwissProt search result: AK243212 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK243212 J100042M20 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 2e-25 ...

  9. SwissProt search result: AK106011 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106011 001-206-B02 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 4e-49 ...

  10. SwissProt search result: AK099308 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK099308 J023144C09 (P00786) Cathepsin H precursor (EC 3.4.22.16) (Cathepsin B3) (Cat...hepsin BA) [Contains: Cathepsin H mini chain; Cathepsin H heavy chain; Cathepsin H light chain] CATH_RAT 2e-47 ...

  11. Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L

    OpenAIRE

    1996-01-01

    The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent compl...

  12. Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

    OpenAIRE

    Yasothornsrikul, Sukkid; Greenbaum, Doron; Medzihradszky, Katalin F.; Toneff, Thomas; Bundey, Richard; Miller, Ruthellen; Schilling, Birgit; Petermann, Ivonne; Dehnert, Jessica; Logvinova, Anna; Goldsmith, Paul; Neveu, John M.; Lane, William S.; Gibson, Bradford; Reinheckel, Thomas

    2003-01-01

    Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological pro...

  13. Removal of the glycosylphosphatidylinositol anchor from PrPSc by cathepsin D does not reduce prion infectivity

    OpenAIRE

    Lewis, P. A.; Properzi, F.; Prodromidou, K.; Clarke, A R; Collinge, J.; Jackson, G S

    2006-01-01

    According to the protein-only hypothesis of prion propagation, prions are composed principally of PrPSc, an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrPC), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrPSc to the activity of PI-PLC (phosphoinositide specific phospholipase C), an e...

  14. Localisation of a gene for prepubertal periodontitis to chromosome 11q14 and identification of a cathepsin C gene mutation

    OpenAIRE

    Hart, T; Hart, P; Michalec, M; Zhang, Y.; Marazita, M; Cooper, M.; Yassin, O; Nusier, M; Walker, S

    2000-01-01

    Prepubertal periodontitis (PPP) is a rare and rapidly progressive disease of young children that results in destruction of the periodontal support of the primary dentition. The condition may occur as part of a recognised syndrome or may occur as an isolated finding. Both autosomal dominant and recessive forms of Mendelian transmission have been reported for PPP. We report a consanguineous Jordanian family with four members affected by PPP in two nuclear sibships. The parents of the affected s...

  15. Contribution of cathepsins B, L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Godiksen, Helene; Morzel, M.; Hyldig, Grethe; Jessen, Flemming

    2009-01-01

    Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days oil ice were obtained by SDS-PAGE. The link between protein hand intensities and firmness of trout fillets was examined through a correlation...

  16. Development of a chitinase and v-cathepsin negative bacmid for improved integrity of secreted recombinant proteins

    NARCIS (Netherlands)

    Kaba, S.A.; Salcedo, A.M.; Wafula, P.O.; Vlak, J.M.; Oers, van M.M.

    2004-01-01

    The application of the baculovirus-in sect cell expression system for the production of integral membrane and secreted proteins is often more troublesome than for cytoplasmic proteins. One protein expressed at low levels in insect cells is the Theileria parva sporozoite surface protein p67. Theileri

  17. Cathepsin K gene mutations and 1q21 haplotypes in at patients with pycnodysostosis in an outbred population

    DEFF Research Database (Denmark)

    Haagerup, Annette; Hertz, Jens Michael; Christensen, M F;

    2000-01-01

    : a one base transition in exon8, c926T > C, causing a single amino acid substitution leucine-->proline, L309P; A 3' splice site mutation in intron 2, c121-1G > A, causing deletion of all exon 3, 41V-81Mdel; and the exon 3 missense mutation c236G > A leading to residue G79E. In three of the families...... disease locus was common to all the patients. This haplotype was found on seven chromosomes identical by state, IBS, out of the possible eight carrying the 926T > C mutation. Founder effect, locus homogeneity, and allele heterogeneity regarding pycnodysostosis within this population are discussed. Finally...

  18. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    OpenAIRE

    Bibiana Gonzales Santana; Dalton, John P.; Fabio Vasquez Camargo; Michael Parkinson; Momar Ndao

    2013-01-01

    BACKGROUND: Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, the...

  19. Proteomic Analysis of Circulating Monocytes Identifies Cathepsin D as A Potential Novel Plasma Marker of Acute Coronary Syndromes

    OpenAIRE

    Fernando Vivanco; Jesús Egido; José Tuñón; Lorenzo Lopez-Bescos; Gloria Alvarez-Llamas; Julio Jiménez-Narcher; Luis Miguel Blanco-Colio; Jose Luis Martin-Ventura; Fernando de la Cuesta; Verónica M. Dardé; Maria G Barderas

    2008-01-01

    We have performed a proteomic analysis of peripheral blood monocytes from ACS patients in comparison with healthy subjects and stable coronary patients in order to search novel biomarkers of ACS in circulating monocytes. Monocytes were isolated from blood of patients with non-ST elevation ACS (n = 27) at day 0, 2 and 6 months, and from patients with stable coronary disease (n = 10) and matched healthy controls (n = 11). The proteomic analysis of monocytes from ACS patients at day 0 showed ...

  20. Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins

    OpenAIRE

    Sciani, Juliana Mozer; Antoniazzi, Marta Maria; Neves, Adriana da Costa; Pimenta, Daniel Carvalho

    2013-01-01

    Background Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in ord...

  1. Activation Route of the Schistosoma mansoni Cathepsin B1 Drug Target: Structural Map with a Glycosaminoglycan Switch

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Horn, Martin; Řezáčová, Pavlína; Marešová, Lucie; Fajtová, Pavla; Brynda, Jiří; Vondrášek, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2014-01-01

    Roč. 22, č. 12 (2014), s. 1786-1798. ISSN 0969-2126 R&D Projects: GA ČR GA203/09/1585; GA MŠk LH12023; GA MŠk LO1302 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : human procathepsin B * slow binding inhibition * asparaginyl endopeptidase Subject RIV: CE - Biochemistry Impact factor: 5.618, year: 2014

  2. Propeptide of cathepsin B1 from human blood fluke: structure-function mapping and small mimetics design

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Jílková, Adéla; Vondrášek, Jiří; Caffrey, C. R.; Mareš, Michael

    Portorož : -, 2010. s. 72-72. [Symposium on Proteases , Inhibitors and Biological Control /12./. 25.9.-29.9.2010, Portorož] R&D Projects: GA ČR GA203/09/1585 Institutional research plan: CEZ:AV0Z40550506 Keywords : protease * schistosoma * propeptide Subject RIV: CE - Biochemistry

  3. Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1 is involved in host gut penetration

    Directory of Open Access Journals (Sweden)

    Long Ying

    2015-01-01

    Full Text Available Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

  4. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid;

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc...

  5. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

    Directory of Open Access Journals (Sweden)

    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  6. c-Myb regulates matrix metalloproteinases 1/9, and cathepsin D: implications for matrix-dependent breast cancer cell invasion and metastasis

    Czech Academy of Sciences Publication Activity Database

    Knopfová, L.; Beneš, P.; Pekarčíková, L.; Hermanová, M.; Masařík, M.; Pernicová, Zuzana; Souček, Karel; Šmarda, J.

    2012-01-01

    Roč. 11, MAR 23 (2012), ID 15. ISSN 1476-4598 R&D Projects: GA MZd NS9600 Grant ostatní: GA AV ČR(CZ) IAA501630901 Institutional support: RVO:68081707 Keywords : c-Myb * Metastasis * Breast cancer Subject RIV: BO - Biophysics Impact factor: 5.134, year: 2012

  7. GX15-070 (obatoclax) induces apoptosis and inhibits cathepsin D and L mediated autophagosomal lysis in antiestrogen resistant breast cancer cells

    OpenAIRE

    Schwartz-Roberts, Jessica L; Shajahan, Ayesha N; Cook, Katherine L.; Wärri, Anni; Abu-Asab, Mones; Clarke, Robert

    2013-01-01

    In estrogen receptor positive (ER+) breast cancer cells, BCL2 overexpression contributes to antiestrogen resistance. Direct targeting of the antiapoptotic BCL2 members with GX15-070 (GX; obatoclax), a BH3-mimetic currently in clinical development, is an attractive strategy to overcome antiestrogen resistance in some breast cancers. Recently, GX has been shown to induce both apoptosis and autophagy, yet the underlying cell death mechanisms have yet to be elucidated. Here we show that GX is mor...

  8. Characterization of a recombinant Cathepsin B-Like cysteine peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A putative target control of citrus huanglongbing

    Science.gov (United States)

    Huanglongbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) spread by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Among the control strategies for H...

  9. Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the parasite cysteine proteases rhodesain, cruzain, and Schistosoma mansoni cathepsin B1.

    Science.gov (United States)

    Fonseca, Nayara Cristina; da Cruz, Luana Faria; da Silva Villela, Filipe; do Nascimento Pereira, Glaécia Aparecida; de Siqueira-Neto, Jair Lage; Kellar, Danielle; Suzuki, Brian M; Ray, Debalina; de Souza, Thiago Belarmino; Alves, Ricardo José; Sales Júnior, Policarpo Ademar; Romanha, Alvaro José; Murta, Silvane Maria Fonseca; McKerrow, James H; Caffrey, Conor R; de Oliveira, Renata Barbosa; Ferreira, Rafaela Salgado

    2015-05-01

    The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 μM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 μM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series. PMID:25712353

  10. Investigation of two novel biochemical markers of inflammation, matrix metalloproteinase and cathepsin generated fragments of C-reactive protein, in patients with ankylosing spondylitis

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Schett, Georg; Zhang, Chen;

    2012-01-01

    prognostic marker for AS has not provided the sought accuracy and specificity. We hypothesized that local enzymatic activity in the disease-affected tissue, which is associated with extensive tissue turnover may, by cleavage, modify the CRP produced in the liver. These cleavage products may provide...

  11. A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

    OpenAIRE

    Deretic, V; Bewley, M. A.; Marriott, H. M.; Tulone, C.; Francis, S E; Mitchell, T J; Read, R. C.; Chain, B.; Kroemer, G; Whyte, M.K.B.; Dockrell, D. H.

    2011-01-01

    Author Summary Tissue macrophages frequently undergo a program of cell death, termed apoptosis, following sustained ingestion and killing of bacteria. In macrophages, induction of apoptosis enhances bacterial killing when macrophages' initial killing capacity is exhausted. We have investigated the mechanism of apoptosis in macrophages exposed to pneumococci, the commonest cause of bacterial pneumonia. We show that the cell structure containing ingested bacteria, the phagolysosome, becomes per...

  12. New approach for osteoporosis treatment: cathepsin K inhibitor, Odanacatib%组织蛋白酶K抑制剂Odanacatib的研究进展

    Institute of Scientific and Technical Information of China (English)

    崔敏; 于灵芝

    2011-01-01

    组织蛋白酶K是一种在破骨细胞高表达的溶酶体蛋白酶,在骨胶原的降解过程中发挥关键作用.临床前研究证实,组织蛋白酶K抑制剂可以逆转去卵巢动物的骨量流失,恢复其骨强度.Odanacatib是一种高度选择性的组织蛋白酶K抑制剂,几乎不产生硬斑病样皮肤病变.一项为期3年、对绝经后骨量减少或骨质疏松症的临床研究发现,较之安慰剂组,Odanacatib治疗组的骨密度明显升高,治疗效果与目前广泛应用的双膦酸盐等抗吸收药物相当,除此之外还具有不抑制骨形成的优点.

  13. Cathepsin-L and Transglutaminase Dependent Processing of ps20: A novel Mechanism for ps20 Regulation via ECM Cross-Linking

    OpenAIRE

    Hickman, Oliver J; Dasgupta, Prokar; Galustian, Christine; Smith, Richard A; Vyakarnam, Annapurna

    2016-01-01

    Whey-acidic-protein (WAP) four-disulphide core (WFDC) proteins have important roles in the regulation of innate immunity, anti-microbial function, and the inhibition of inflammatory proteases at mucosal surfaces. It was recently demonstrated that the WFDC protein, prostate stromal 20 (ps20), encoded by the WFDC1 gene, is a potent growth inhibitory factor, and shares with other WFDC proteins the ability to modulate wound healing processes and immune responses to viral infections. However, ps20...

  14. Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment.

    Science.gov (United States)

    Tichy, Elisia D; Stephan, Zachary A; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J

    2013-05-01

    Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs. PMID:23500643

  15. Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing

    OpenAIRE

    Saghizadeh, Mehrnoosh; Epifantseva, Irina; Hemmati, David M.; Ghiam, Chantelle A.; Brunken, William J; Ljubimov, Alexander V

    2013-01-01

    Adenovirus-driven shRNA silencing of select proteinases upregulated in diabetic corneas restored normal wound healing time, the expression of diabetes-altered markers including limbal stem cell markers, and patterns of activated EGFR and Akt in human diabetic corneal organ cultures. The maximum effect was obtained combining proteinase shRNA with c-met overexpression.

  16. Promising markers for the detection of premature senescence tumor cells induced by ionizing radiation: Cathepsin D and eukaryotic translation elongation factor 1

    International Nuclear Information System (INIS)

    Recently, it has been proved that induction of senescence could be a promising way of tumor treatment. Senescence was originally described in normal human cells undergoing a finite number of divisions before permanent growth arrest. It has now become regarded more broadly as a general biological program of terminal growth arrest. A variety of stresses such as ionizing radiation (IR), oxidative stress, oncogenic transformation, DNA damaging agents triggers stress-induced premature senescence, i.e. rapid and permanent cell growth arrest. Therefore, premature senescence is bona fide barrier to tumorigenesis and hallmark of premalignant tumors. However, there is lack of obvious markers for senescent tumor cells. To identify useful premature senescence markers for tumor cells, we monitored the changes of protein expression profile in IR-induced premature senescence MCF7 human breast cancer cells. We identified biomarkers which evidently changed their expression levels in ionizing radiation-induced senescenct tumor cells

  17. Imunolokalizace trávicích peptidáz v nymfách klíštěte \\kur{Ixodes ricinus} v průběhu sání na hostiteli a během přeměny na dospělce

    OpenAIRE

    SCHRENKOVÁ, Jana

    2011-01-01

    Morphology changes in the gut structure of Ixodes ricinus nymphs during blood feeding and metamorphosis to adult was studied by light microscopy. Indirect immunofluorescence was used to localize digestive peptidases (cathepsin B and cathepsin L) during this process.

  18. Odanacatib, a New Drug for the Treatment of Osteoporosis: Review of the Results in Postmenopausal Women

    OpenAIRE

    José Luis Pérez-Castrillón; Florentino Pinacho; Daniel De Luis; María Lopez-Menendez; Antonio Dueñas Laita

    2010-01-01

    Osteoclasts are specialized cells that initiate the process of bone resorption, which has two phases, dissolution of the mineral component and degradation of the organic matrix, in which cathepsin K plays a key role. Cathepsin K inhibitors, which block the activity of cathepsin on bone resorption lacunae, may be a new therapeutic option in osteoporosis. Odanacatib is a nonpeptidic biaryl inhibitor of cathepsin K. Two studies have evaluated the efficacy and safety of odanacatib, a phase I stud...

  19. Molecular profiling of proteolytic and lectin transcripts in Homalodisca vitripennis (Hemiptera: Auchenorrhyncha: Cicadellidae) feeding on sunflower and cowpea

    Science.gov (United States)

    Homalodisca vitripennis Germar 1821 (Hemiptera: Cicadellidae) (Takiya et al. 2006, syn. H. coagulata (Say)) gut and salivary gland EST libraries were used to isolate cDNA fragments of the genes encoding for cathepsin L, asparaginyl endopeptidase, cathepsin B, metalloendopeptidase, cathepsin D, multi...

  20. Erosive arthritis in a patient with pycnodysostosis: An experiment of nature

    NARCIS (Netherlands)

    Ainola, M.; Valleala, H.; Nykänen, P.; Risteli, J.; Hanemaaijer, R.; Konttinen, Y.T.

    2008-01-01

    Objective. The excellent poster painter Henri de Toulouse-Lautrec is the most famous patient with cathepsin K-deficient pycnodysostosis. Cathepsin K is believed to play a major role in osteoclast-driven bone resorption. In this study we explored the role of cathepsin K in bone resorption in a patien

  1. SNP Analysis of the Cathepsin L Gene in Litopenaeus vannamei%凡纳滨对虾组织蛋白酶L基因的单核苷酸多态性分析

    Institute of Scientific and Technical Information of China (English)

    马宁; 陈晓汉; 曾地刚; 彭敏; 李咏梅

    2008-01-01

    采用PCR产物直接测序法,对凡纳滨对虾40份DNA样本的组织蛋白酶L基因进行单核苷酸多态性(SNP)检测.经过对测序结果统计分析,共发现SNP 20个,分别是:A150C,T226G,G240A,C429T,T453C,G537A,C597T,C645T,G798C,C831T,C955T,C963T,C977T,C982T,G1001C,A1005T,C1117T,C1132T,G1367A,T1391C.其中6个SNP是外显子中的错义突变,3个SNP是外显子中的同义突变,11个SNP是内含子中的突变.本检测为进一步进行凡纳滨对虾生长性状关联研究提供了有用信息.

  2. The prokaryotic expression and biological specialities of Araneus Ventricousus cathepsin B-like gene Avg 1%大腹园蛛Avg 1的原核表达及生物学活性

    Institute of Scientific and Technical Information of China (English)

    卢士英; 任洪林; 潘风光; 孟宪梅; 柳增善

    2007-01-01

    [目的]研究大腹园蛛组织蛋白酶B相关基因Avg 1(基因登录号:AY 302573)编码蛋白的生物学特性.[方法]设计合成引物(AvgS,AvgX),扩增Avg 1,去除信号肽部分基因Avg,将其与原核表达载体pET-28 a(+),pET-20 b连接,转化到E.coli BL 21(DE 3)中.[结果]鉴定为阳性克隆后,用IPTG诱导表达,SDS-PAGE电泳显示Avg-28 a-DE 3在近35.7 ku处出现明显的表达带,其大小与预计分子质量(35.2 ku)相当;经薄层扫描分析显示,表达蛋白量占菌体蛋白总量的39.6%,且表达形式主要为包涵体,而Avg-20 b-DE 3表达产物未见表达带,但RT-PCR方法检测到了Avg mRNA的转录;将表达的包涵体蛋白进行变性及复性处理,与诱导的Avg-20 b-DE 3表达菌经超声波碎菌处理上清液一同进行蛋白酶活性测定,呈复性的包涵体蛋白不具有酶活性,而Avg-20 b-DE 3的碎菌上清液显示明显的蛋白酶活性.表达的包涵体蛋白经电洗脱纯化后,常规方法免疫吉绒獭兔,抗血清经ELISA法检测,呈明显阳性反应,经Western blot检测在35.7 ku处出现特异结合带.[结论]Avg 1原核表达蛋白具有良好的免疫原性.

  3. Homology modeling and SAR analysis of Schistosoma japonicum cathepsin D (SjCD) with statin inhibitors identify a unique active site steric barrier with potential for the design of specific inhibitors

    Czech Academy of Sciences Publication Activity Database

    Caffrey, C. R.; Plachá, L.; Bařinka, Cyril; Hradilek, Martin; Dostál, Jiří; Sajid, M.; McKerrow, J. H.; Majer, P.; Konvalinka, Jan; Vondrášek, Jiří

    2005-01-01

    Roč. 386, č. 4 (2005), 339-349. ISSN 1431-6730 R&D Projects: GA MŠk(CZ) LC512; GA ČR(CZ) GP203/02/P095; GA AV ČR(CZ) IAA400550510 Grant ostatní: SFSF(US) AI 53247; NATO(XE) CLG 974914 Institutional research plan: CEZ:AV0Z4055905 Keywords : schistotoma * proteases of parasites * modelling Subject RIV: CE - Biochemistry Impact factor: 2.577, year: 2005

  4. Effect of some factors used to the chicken meat preservation and processing on the protease activity

    International Nuclear Information System (INIS)

    The obtained results indicated that the cathepsin activity was higher by about 60% in the extract from thigh than from breast muscles. Freezing and defrosting (not stored) of chicken meat did not influence the breast muscle cathepsin activity while they caused a decrease of activity of about 20% in the case of thigh muscles. The increase in cathepsin activity was noticed in both kinds of muscles during storage at -20 °C up to 4 months (45.6% and 19.4% for thigh and breast muscles respectively). The activity of cathepsin in extract from 5 months stored meat reached 80% in case of breast muscles and 83% in case of thigh muscles in relation to control sample respectively. The cathepsin activity significantly increased during heating of breast muscles up to 60 °C, but in case of thigh muscles it was slightly higher than at 50°C. The heating of cured chicken breast muscles up to 60°C caused a non significant growth in cathepsin activity opposite to raw muscles. The cathepsin activity from all cured samples heated up to 70°C were several times lower in relation to control samples. The cathepsin activity of both thigh and breast muscles were resistant to gamma radiation. The investigated factors caused changes in the activity of cathepsin but none of them caused its total inactivation. The changes of cathepsin activity depended on the kind of muscles and the kind and the value of acting factors

  5. Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity▿

    OpenAIRE

    Kaletsky, Rachel L.; Simmons, Graham; Bates, Paul

    2007-01-01

    Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increase...

  6. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    OpenAIRE

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants k...

  7. Dicty_cDB: CHC638 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ) Artemia franciscana cathepsin L-li... 172 8e-42 DQ474246_1( DQ474246 |pid:none) Lygus lineolaris cathepsin...-L mRNA,... 172 8e-42 AY795054_1( AY795054 |pid:none) Artemia franciscana cathepsin ...69 |S58669.1 Entamoeba histolytica cysteine proteinase precursor (ACP1) gene, partial cds. 54 2e-06 2 AZ5471...L pr... 172 8e-42 AY363263_1( AY363263 |pid:none) Triatoma infestans cathepsin L-...307 |M27307.1 Entamoeba histolytica cysteine protease gene, partial cds. 54 0.003 1 BQ834906 |BQ834906.1 Po_

  8. Dicty_cDB: SFI542 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available AF326781.1 Triticum monococcum actin (ACT-1) gene, partial cds; putative chromosome condensation factor (CCF...cidon perlevis cathepsin L ... 163 3e-39 AF147207_1( AF147207 |pid:none) Artemia franciscana cathepsin...3e-39 AY795054_1( AY795054 |pid:none) Artemia franciscana cathepsin L pr... 163 3... L-li... 163 3e-39 AY363263_1( AY363263 |pid:none) Triatoma infestans cathepsin L-lik... 163 ...c 28.0 %: nuclear 12.0 %: Golgi 8.0 %: mitochondrial 4.0 %: cytoskeletal 4.0 %: vesicles of secretory system >> predict

  9. Chemoenzymatic synthesis of organoselenium(IV) compounds and their evaluation as cysteine protease inhibitors

    International Nuclear Information System (INIS)

    A series of organoselenium dihalides (organoselenanes) was synthesized from organoselenides using a chemoenzymatic approach. The organoselenanes have variations in their stereochemistry and in the halogen atom bonded to the selenium atom. Because of the unique selenium-thiol chemistry displayed by several organoselenium compounds, the organoselenanes were evaluated as new potential inhibitors of cysteine proteases (cathepsins S and V). By the analysis of the second-order rate constants of the inhibition of cathepsin S and V, it was possible to conclude that organoselenanes inhibited the cathepsin S faster than cathepsin V. It was observed higher inhibitory potencies for the dibromo organoselenanes derivatives than the dichloro analogues. In addition, the present data suggest the use of hypervalent selenium compounds as novel motifs for cysteine proteases inhibitors. (author)

  10. Chemoenzymatic synthesis of organoselenium(IV) compounds and their evaluation as cysteine protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Piovan, Leandro; Andrade, Leandro H., E-mail: leandroh@iq.usp.b [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica; Alves, Marcio F.M.; Juliano, Luiz; Cunha, Rodrigo L.O.R, E-mail: rodrigo.cunha@ufabc.edu.b [Universidade Federal de Sao Paulo (Unifesp/EPM), Sao Paulo, SP (Brazil). Dept. de Biofisica; Broemme, Dieter [University of British Columbia, Vancouver (Canada). Dept. of Dentistry

    2010-07-01

    A series of organoselenium dihalides (organoselenanes) was synthesized from organoselenides using a chemoenzymatic approach. The organoselenanes have variations in their stereochemistry and in the halogen atom bonded to the selenium atom. Because of the unique selenium-thiol chemistry displayed by several organoselenium compounds, the organoselenanes were evaluated as new potential inhibitors of cysteine proteases (cathepsins S and V). By the analysis of the second-order rate constants of the inhibition of cathepsin S and V, it was possible to conclude that organoselenanes inhibited the cathepsin S faster than cathepsin V. It was observed higher inhibitory potencies for the dibromo organoselenanes derivatives than the dichloro analogues. In addition, the present data suggest the use of hypervalent selenium compounds as novel motifs for cysteine proteases inhibitors. (author)

  11. ALCALOIDES ACRIDÔNICOS INIBEM CATEPSINA L E V

    Directory of Open Access Journals (Sweden)

    Emerson F. Marques

    2016-01-01

    Full Text Available Cathepsins represent a class of enzymes that has the primary function of randomly degrading proteins in the lysosomes, although are also involved in different pathologies. The aim of this paper was to evaluate the capacity of acridone alkaloids isolated from Swinglea glutinosa (Rutaceae to inhibit cathepsin L in vitro . The IC50 values found were in the 0.8-57 µM range and the most promising compounds were alkaloids 1 and 2, with IC50 of 0.9 and 0.8 µM, respectively. Enzyme kinetics revealed that they are reversible competitive inhibitors with respect to the substrate Z-FR-MCA. This small series of acridone alkaloids showed low selectivity for both cathepsins, but represent promising lead candidates for the further development of competitive cathepsin L and V inhibitors.

  12. AcEST: DK957313 [AcEST

    Lifescience Database Archive (English)

    Full Text Available epsin L1 OS=Canis familiaris GN=CTSL1 ... 59 1e-08 sp|Q5E998|CATL2_BOVIN Cath...epsin B OS=Nicotiana benthamiana GN=C... 226 9e-58 tr|Q40413|Q40413_NICRU Cathepsin B...FA Sbjct: 249 YKNGPVEVSFTVYEDFA 265 >tr|Q1HER6|Q1HER6_NICBE Cathepsin B OS=Nicotiana benthamiana GN=Cath...F 259 >sp|P43509|CPR5_CAEEL Cathepsin B-like cysteine proteinase 5 OS=Caenorhabditis elegan... Peptidase C1A, papain; Somatotropin hormo... 214 3e-54 tr|Q93VC9|Q93VC9_ARATH At1g02300/T6A9_10 OS=Arabidopsis thalian

  13. AcEST: DK962994 [AcEST

    Lifescience Database Archive (English)

    Full Text Available =1 SV=1 101 3e-21 sp|Q9WGE0|CATV_NPVHC Viral cathepsin OS=Hyphantria cunea nuclea...sult : Swiss-Prot sp_hit_id P43296 Definition sp|P43296|RD19A_ARATH Cysteine proteinase RD19a OS=Arabidopsis thaliana Align length...epsin L OS=Schistosoma mansoni GN=CL1 P... 124 4e-28 sp|Q9R013|CATF_MOUSE Cathepsin F OS=Mus musculus GN...31 GE GYY + RG CGVNTM S+ Sbjct: 458 GEKGYYYLHRGSGACGVNTMASS 480 >sp|Q26534|CATL_SCHMA Cathepsin L OS=Schistosoma mansoni...P_TRYBB Cysteine proteinase OS=Trypanosoma brucei b... 105 2e-22 sp|P06797|CATL1_MOUSE Cathepsin L1 OS=Mus m

  14. On How Mammary Gland Reprogramming Metalloproteinases Couple Form with Function

    OpenAIRE

    Sloane, Bonnie F

    2012-01-01

    Metalloproteinases in extracellular proteolytic pathways are critical to mammary gland biology and tumorigenesis. However, intracellular and membrane proteases (e.g., caspases and cathepsins) may also play important roles.

  15. Neuroimmune regulation of alcohol consumption: Behavioral validation of genes obtained from genomic studies

    OpenAIRE

    Blednov, Yuri A; Ponomarev, Igor; Geil, Chelsea; Bergeson, Susan; Koob, George F.; Harris, R. Adron

    2011-01-01

    Analysis of mouse brain gene expression, using strains that differ in alcohol consumption, provided a number of novel candidate genes that potentially regulate alcohol consumption. We selected six genes [beta-2-microglobulin (B2m), cathepsin S (Ctss), cathepsin F (Ctsf), interleukin 1 receptor antagonist (Il1rn), CD14 molecule (Cd14) and interleukin 6 (Il6)] for behavioral validation using null mutant mice. These genes are known to be important for immune responses but were not specifically l...

  16. Proteases of neutrophilic granulocytes

    Directory of Open Access Journals (Sweden)

    Wiesława Roszkowska-Jakimiec

    2002-06-01

    Full Text Available The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B, serine proteases (granulocytic elastase, cathepsin G, protease 3, membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine, cysteine and aspartic cathepsins. The role of these proteases in the pathology and diagnostics of certain diseases was considered.

  17. Gold-containing drugs and the control of proteolytic enzymes.

    OpenAIRE

    Rohozková, D.; Steven, F. S.

    1983-01-01

    1 NaAuCl4 and aurothioglucose inhibited trypsin in free solution without the need of a carrier molecule. 2 NaAuCl4, aurothioglucose, aurothiomalate, auranofin and chloro-triethyl phosphine) gold all inhibited the trypsin-like neutral protease on the surfaces of Ehrlich ascites tumour cell membranes equally well. 3 Crude cathepsin preparations were activated by low concentrations of dithiothreitol and also by aurothioglucose, due to the displacement of an inhibitor. 4 Thiol-activated cathepsin...

  18. Discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and TbcatB

    OpenAIRE

    Mallari, Jeremy P.; Shelat, Anang; Kosinski, Aaron; Conor R Caffrey; Connelly, Michele; Zhu, Fangyi; McKerrow, James H.; Guy, R. Kiplin

    2008-01-01

    Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases of T.brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention. Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured T.bruc...

  19. The dynamics of cysteine proteinase activity in brain structures of irrigated rat descendants during ontogenetic development

    OpenAIRE

    Чорна, Валентина Іванівна; Лянна, Ольга Леонідівна

    2016-01-01

    The aim of the work was to investigate the kind of lysosomal cysteine cathepsin L activity dependency in brain structures of irradiated rat descendants during ontogenetic development. It was shown that fractional x-ray radiation (25 cGy) of the female rats induced different changes of cathepsin L activity levels and their redistribution in brain structures of female rats’ descendants during postnatal development with the advantages of nonsedimentational activity that had maximum at the 6th da...

  20. Venomous protease of aphid soldier for colony defense

    OpenAIRE

    Kutsukake, Mayako; Shibao, Harunobu; Nikoh, Naruo; Morioka, Mizue; Tamura, Tomohiro; Hoshino, Tamotsu; Ohgiya, Satoru; Fukatsu, Takema

    2004-01-01

    In social aphids, morphological, behavioral, and physiological differences between soldiers and normal insects are attributed to differences in gene expression between them, because they are clonal offspring parthenogenetically produced by the same mothers. By using cDNA subtraction, we identified a soldier-specific cysteine protease of the family cathepsin B in a social aphid, Tuberaphis styraci, with a second-instar soldier caste. The cathepsin B gene was specifically expressed in soldiers ...

  1. Atorvastatin hemmer aktiviteten av cysteinproteaser og stimulerer sekresjonen av MMP-9

    OpenAIRE

    2012-01-01

    I tillegg til de etablerte kolesterolsenkende effektene av statiner, indikerer nye studier at statiner utviser tilleggseffekter (pleiotrope effekter) som kan forklare deres terapeutiske suksess. Nylig ble det vist at mRNA for cysteinproteasene legumain og cathepsin B ble nedregulert i monocytter/makrofager hos pasienter behandlet med atorvastatin. Makrofager spiller en sentral rolle i utviklingen av aterosklerotiske plakk, og det er nylig gjort funn som viser at legumain og cathepsin B er opp...

  2. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    OpenAIRE

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R; Simmons, Graham

    2015-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coron...

  3. Odanacatib for the treatment of postmenopausal osteoporosis: development history and design and participant characteristics of LOFT, the Long-Term Odanacatib Fracture Trial

    OpenAIRE

    Bone, H. G.; Dempster, D.W.; Eisman, J. A.; Greenspan, S. L.; McClung, M. R.; Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan); Papapoulos, S; Shih, W. J.; Rybak-Feiglin, A.; Santora, A. C.; Verbruggen, N.; Leung, A. T.; Lombardi, A.

    2014-01-01

    Summary Odanacatib is a cathepsin K inhibitor investigated for the treatment of postmenopausal osteoporosis. Phase 2 data indicate that 50 mg once weekly inhibits bone resorption and increases bone mineral density, with only a transient decrease in bone formation. We describe the background, design and participant characteristics for the phase 3 registration trial. Introduction Odanacatib (ODN) is a selective cathepsin K inhibitor being evaluated for the treatment of osteoporosis. In a phase ...

  4. Cellular and molecular mechanisms of osteoporosis: current concepts and future direction treatment

    OpenAIRE

    A. T. Dolzhenko; S. Sagalovsky

    2016-01-01

    The article presents review of literature dedicated to the contemporary view on the cellular-molecular mechanisms of the bone remodeling and pathogenesis of the osteoporosis. The discovery of the cytokine RANKL-RANK-OPG system and significant role of the cathepsin K in process bone remodeling has made progress in understanding the mechanisms development disease and possible to development drugs of the new generation – denosumab, a fully human RANKL monoclonal antibody and inhibitor cathepsin ...

  5. Potential role of odanacatib in the treatment of osteoporosis

    OpenAIRE

    Ng KW

    2012-01-01

    Kong Wah NgDepartment of Endocrinology and Diabetes and St Vincent’s Institute, St Vincent’s Hospital, Fitzroy, Victoria, AustraliaAbstract: Cathepsin K is a key enzyme involved in the degradation of organic bone matrix by osteoclasts. Inhibition of bone resorption observed in human and animal models deficient for cathepsin K has identified this enzyme as a suitable target for intervention by small molecules with the potential to be used as therapeutic agents in the treatm...

  6. In Vitro and In Vivo Imaging of Peptide-Encapsulated Polymer Nanoparticles for Cancer Biomarker Activated Drug Delivery

    OpenAIRE

    Kulsharova, Gulsim K.; Lee, Matthew B; Cheng, Felice; Haque, Munima; Choi, Hyungsoo; Kim, Kyekyoon; O’Brien, William D.; Liu, G. Logan

    2013-01-01

    Gelatin nanoparticles coated with Cathepsin D-specific peptides were developed as a vehicle for the targeted delivery of the cancer drug doxorubicin (DOX) to treat breast malignancy. Cathepsin D, a breast cancer cell secretion enzyme, triggered the release of DOX by digesting the protective peptide-coating layer of nanoparticles. Fabricated nanoparticles were successfully detected with ultrasound imaging in both in vitro conditions and in vivo mouse cancer models. Cell viability experiments w...

  7. CA-074Me compound inhibits osteoclastogenesis via suppression of the NFATc1 and c-FOS signaling pathways.

    Science.gov (United States)

    Patel, Neel; Nizami, Saqib; Song, Lee; Mikami, Maya; Hsu, Anny; Hickernell, Thomas; Chandhanayingyong, Chandhanarat; Rho, Shim; Compton, Jocelyn T; Caldwell, Jon-Michael; Kaiser, Philip B; Bai, Hanying; Lee, Heon Goo; Fischer, Charla R; Lee, Francis Y

    2015-10-01

    The osteoclast is an integral cell of bone resorption. Since osteolytic disorders hinge on the function and dysfunction of the osteoclast, understanding osteoclast biology is fundamental to designing new therapies that curb osteolytic disorders. The identification and study of lysosomal proteases, such as cathepsins, have shed light on mechanisms of bone resorption. For example, Cathepsin K has already been identified as a collagen degradation protease produced by mature osteoclasts with high activity in the acidic osteoclast resorption pits. Delving into the mechanisms of cathepsins and other osteoclast related compounds provides new targets to explore in osteoclast biology. Through our anti-osteoclastogenic compound screening experiments we encountered a modified version of the Cathepsin B inhibitor CA-074: the cell membrane-permeable CA-074Me (L-3-trans-(Propylcarbamoyl) oxirane-2-carbonyl]-L-isoleucyl-L-proline Methyl Ester). Here we confirm that CA-074Me inhibits osteoclastogenesis in vivo and in vitro in a dose-dependent manner. However, Cathepsin B knockout mice exhibited unaltered osteoclastogenesis, suggesting a more complicated mechanism of action than Cathepsin B inhibition. We found that CA-074Me exerts its osteoclastogenic effect within 24 h of osteoclastogenesis stimulation by suppression of c-FOS and NFATc1 pathways. PMID:25428830

  8. Proteinases and associated genes of parasitic helminths.

    Science.gov (United States)

    Tort, J; Brindley, P J; Knox, D; Wolfe, K H; Dalton, J P

    1999-01-01

    Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus. By far the largest number of papers in the literature and entries to the sequence data bases dealing with proteinases of parasitic helminths report on enzymes belonging to the papain superfamily of cysteine proteinases. Accordingly, the final section of the review is devoted to a phylogenetic analysis of this superfamily using over 150 published sequences. This analysis shows that the papain superfamily can be divided into two major branches. Branch A contains the cathepin Bs, the cathepsin Cs and a novel family termed cathepsin Xs, while Branch B contains the cruzipains

  9. Regulatory elements within the prodomain of Falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Kailash C Pandey

    Full Text Available Falcipain-2, a papain family cysteine protease of the malaria parasite Plasmodium falciparum, plays a key role in parasite hydrolysis of hemoglobin and is a potential chemotherapeutic target. As with many proteases, falcipain-2 is synthesized as a zymogen, and the prodomain inhibits activity of the mature enzyme. To investigate the mechanism of regulation of falcipain-2 by its prodomain, we expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2. We identified a C-terminal segment (Leu(155-Asp(243 of the prodomain, including two motifs (ERFNIN and GNFD that are conserved in cathepsin L sub-family papain family proteases, as the mediator of prodomain inhibitory activity. Circular dichroism analysis showed that the prodomain including the C-terminal segment, but not constructs lacking this segment, was rich in secondary structure, suggesting that the segment plays a crucial role in protein folding. The falcipain-2 prodomain also efficiently inhibited other papain family proteases, including cathepsin K, cathepsin L, cathepsin B, and cruzain, but it did not inhibit cathepsin C or tested proteases of other classes. A structural model of pro-falcipain-2 was constructed by homology modeling based on crystallographic structures of mature falcipain-2, procathepsin K, procathepsin L, and procaricain, offering insights into the nature of the interaction between the prodomain and mature domain of falcipain-2 as well as into the broad specificity of inhibitory activity of the falcipain-2 prodomain.

  10. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  11. Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

    International Nuclear Information System (INIS)

    The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki=3.3 nM) and baupain (Ki=2.1x10-8 M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125 nM), cathepsin K (Ki=0.76 nM), cathepsin L (Ki=0.6 nM), and cathepsin V (Ki=1.0 nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases

  12. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

    Directory of Open Access Journals (Sweden)

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

  13. Antigen expression on recurrent meningioma cells

    International Nuclear Information System (INIS)

    Meningiomas are intracranial brain tumours that frequently recur. Recurrence rates up to 20% in 20 years for benign meningiomas, up to 80% for atypical meningiomas and up to 100% for malignant meningiomas, have been reported. The most important prognostic factors for meningioma recurrence are meningioma grade, meningioma invasiveness and radicality of neurosurgical resection. The aim of our study was to evaluate the differences in antigenic expression on the surface of meningioma cells between recurrent and non-recurrent meningiomas. 19 recurrent meningiomas and 35 non-recurrent meningiomas were compared regarding the expression of MIB-1 antigen, progesterone receptors, cathepsin B and cathepsin L, using immunohistochemistry. MIB-1 antigen expression was higher in the recurrent meningioma group (p=0.001). No difference in progesterone receptor status between recurrent and non-recurrent meningiomas was confirmed. Immunohistochemical intensity scores for cathepsin B (p= 0.007) and cathepsin L (p<0.001) were both higher in the recurrent than in the non-recurrent meningioma group. MIB-1 antigen expression is higher in recurrent compared to non-recurrent meningiomas. There is no difference in expression of progesterone receptors between recurrent and non-recurrent meningiomas. Cathepsins B and L are expressed more in recurrent meningiomas

  14. 探讨M2型丙酮酸激酶和组织蛋白酶D在卵巢浆液性囊腺瘤和交界性浆液性肿瘤组织中的表达情况及意义%Expressions and significance of type M2 pyruvate kinase and cathepsin D in ovarian serous cystadenoma and ovarian serous borderline tumor

    Institute of Scientific and Technical Information of China (English)

    赵海宁; 施志军; 李冀红

    2015-01-01

    目的 探讨卵巢浆液性囊腺瘤和交界性浆液性肿瘤组织中M2型丙酮酸激酶(M2-PK)和组织蛋白酶D(Cath-D)的表达水平,为肿瘤的诊断、治疗及防止复发提供指导性的意见.方法 采用免疫组织化学法(SP)分别对卵巢浆液性囊腺瘤(对照组)和交界性浆液性肿瘤(观察组)组织中M2-PK、Cath-D的表达水平进行检测,对比两组M2-PK、Cath-D表达的阳性率、灵敏度、特异度.结果 M2-PK在对照组和观察组肿瘤组织表达中阳性率分别为38.00%和72.00%,Cath-D在肿瘤组织表达中阳性率分别为24.00%和66.00%,两组间相比差异有统计学意义(P<0.05);M2-PK蛋白在观察组肿瘤检测中的灵敏度和特异度分别为74.00%和61.54%,Cath-D蛋白分别为66.00%和78.57%.结论 M2-PK和Cath-D蛋白的表达对卵巢交界性浆液性肿瘤早期诊断、治疗及预后有着重要的临床价值.

  15. Occurrence of aspartyl proteases in brine after herring marinating.

    Science.gov (United States)

    Szymczak, Mariusz; Lepczyński, Adam

    2016-03-01

    Herrings are marinated in a brine consisting of salt and acetic acid. During marinating, various nitrogen fractions diffuse from fish flesh to the brine, causing significant nutritional quality losses of the raw material. In this study, it has been demonstrated for the first time that proteases diffuse from the fish to the marinating brine. Using ammonium sulphate precipitation and affinity chromatography on pepstatin-A agarose bed the aspartyl proteases were purified and concentrated over 2600-fold from a marinating brine. Pepstatin-A completely inhibited the activity of the purified preparation. The preparation was active against fluorogenic substrates specific for cathepsin D and E and inactive against substrates specific for cysteine cathepsins. Depending on incubation time, the preparation showed pH-optimum at 2.0 or 4.5. The 2D SDS-PAGE separation demonstrated the presence of a few proteins with molecular weights and pI values typical of cathepsin D, E and pepsin. PMID:26471581

  16. Dicty_cDB: Contig-U16296-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 1e-48 AM409328_1( AM409328 |pid:none) Trifolium pratense partial cp8 gen... 155 1e-48 DQ016552_1( DQ016552 |pid:none) Par...s cathepsin L1, mRNA (c... 160 9e-44 AF498292_1( AF498292 |pid:none) Danio rerio cathepsin mRNA, partia...) Periserrula leucophryna cysteine p... 186 1e-49 EU025855_1( EU025855 |pid:none) Dermacentor variabilis cathepsin...242. 299 1e-76 1 ( AU062230 ) Dictyostelium discoideum slug cDNA, clone SLI528. 210 7e-54 2 ( X85123 ) Artificial sequences clonin...lea x heilbornii VXH-C mRNA, partial cds. 48 2e-09 3 ( EX291630 ) 1579439_5_B05_031 PY06 Carica papaya cDNA,

  17. AcEST: DK956763 [AcEST

    Lifescience Database Archive (English)

    Full Text Available =Mus musculus GN=Ctsd PE=1 SV=1 89 1e-17 sp|Q4LAL9|CATD_CANFA Cathepsin D OS=Cani... sp|P28713|PEPA4_RABIT Pepsin II-4 OS=Oryctolagus cuniculus PE=2 ... 84 5e-16 sp|Q9DEX3|CATD_CLUHA Cathepsin D OS=Clupea ha...25:3389-3402. Query= DK956763|Adiantum capillus-veneris mRNA, clone: TST39A01NGRL0026_J11, 5' (521 letters) Database: uni...epsin D OS=Chionodraco hamatus GN=ctsd ... 90 7e-18 sp|P18242|CATD_MOUSE Cathepsin D OS... sp|P28712|PEPA1_RABIT Pepsin II-1 OS=Oryctolagus cuniculus PE=2 ... 87 4e-17 sp|P80209|CATD_BOVIN Cath

  18. AcEST: DK953744 [AcEST

    Lifescience Database Archive (English)

    Full Text Available lus gallus GN=CTSD PE=1 SV=1 94 5e-19 sp|Q4LAL9|CATD_CANFA Cathepsin D OS=Canis familiaris GN=CTSD PE=... 92...DTGSSNLWVPSVHCHLLDIACLLHHKYDASK 126 >sp|Q4LAL9|CATD_CANFA Cathepsin D OS=Canis familiaris GN=CTSD PE=2 SV=1 Length...cids Res. 25:3389-3402. Query= DK953744|Adiantum capillus-veneris mRNA, clone: TST39A01NGRL0018_J12, 5' (531 letters) Database: uni...epsin D OS=Sus scrofa GN=CTSD PE=1 SV=2 94 5e-19 sp|O93428|CATD_CHIHA Cathepsin D OS=Chionodraco hamat...-17 sp|P28712|PEPA1_RABIT Pepsin II-1 OS=Oryctolagus cuniculus PE=2 ... 87 4e-17 sp|Q9MZS8|CATD_SHEEP Cathep

  19. AcEST: DK954095 [AcEST

    Lifescience Database Archive (English)

    Full Text Available lia GN... 61 5e-09 sp|Q9GL24|CATL1_CANFA Cathepsin L1 OS=Canis familiaris GN=CTSL...core E Sequences producing significant alignments: (bits) Value sp|Q5R6D1|CATB_PONAB Cathepsin B OS=Pongo ab...epsin B OS=Nicotiana benthamiana GN=C... 146 8e-34 tr|B7FJ05|B7FJ05_MEDTR Putative uncharacteriz...ne proteinase OS=Ipomoea batatas GN=CathB PE=2 SV=1 Length = 352 Score = 152 bits (385), Expect = 1e-35 Iden...lease 56.9) Link to BlastX Result : Swiss-Prot sp_hit_id Q5R6D1 Definition sp|Q5R6D1|CATB_PONAB Cathepsin B

  20. AcEST: DK953596 [AcEST

    Lifescience Database Archive (English)

    Full Text Available epsin B-like cysteine proteinase 1 OS=... 56 2e-07 sp|Q9GL24|CATL1_CANFA Cathepsin L1 OS=Canis...epsin B OS=Nicotiana benthamiana GN=C... 129 7e-32 tr|Q94K85|Q94K85_ARATH Putative cath...ECG 609 LS NDLL+CCGF CG Sbjct: 153 LSVNDLLACCGFLCG 167 >tr|B5BQV5|B5BQV5_RAPSA Cathepsin B-like cysteine pro................done Score E Sequences producing significant alignments: (bits) Value sp|Q5R6D1|CATB_PONAB Cath...TCCGIQCG 147 >sp|P43510|CPR6_CAEEL Cathepsin B-like cysteine proteinase 6 OS=Caenorhabditis elegans GN=cpr-6 PE=1 SV=1 Length

  1. Tumor Necrosis Factor-Alpha and Receptor Activator of Nuclear Factor-κB Ligand Augment Human Macrophage Foam-Cell Destruction of Extracellular Matrix Through Protease-Mediated Processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise Korsager; Dziegiel, Morten; Henriksen, Kim; Karsdal, Morten A

    2012-01-01

    Abstract By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a...... into macrophage foam cells and cultured on a type I collagen matrix in the presence of TNF-alpha and RANK-L. Matrix degradation was measured by the cathepsin K-generated C-terminal cross-linked telopeptide of type I collagen (CTX-I) and the MMP-generated carboxyterminal telopeptide of type I collagen...... (ICTP) in supernatants showing that macrophage foam cells secrete MMPs and cathepsin K, resulting in release of ICTP and CTX-I. Stimulation with TNF-alpha increased CTX-I and ICTP dose dependently, with ICTP levels increasing by 59% and CTX-I levels increasing by 43%. RANK-L enhanced the release of CTX...

  2. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas;

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes....... Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated these with the......-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrP. Our results indicate that effects in the ELS...

  3. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  4. Dicty_cDB: AFH148 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available toma infestans cathepsin L-lik... 191 1e-47 EF670319_1( EF670319 |pid:none) Artemia persimilis cathepsin... AF147207_1( AF147207 |pid:none) Artemia franciscana cathepsin L-li... 193 2e-48 AY795054_1( AY795054 |pid:none) Artemia...L (CAT-L1)... 191 8e-48 ( P13277 ) RecName: Full=Digestive cysteine proteinase 1; ... 191 1e-47 AY363263_1( AY363263 |pid:none) Tria...N S58669 |S58669.1 Entamoeba histolytica cysteine proteinase precursor (ACP1) gene, partial cds. 54 2e-06 2 ...c, DNA sequence. 40 0.019 2 AF326781 |AF326781.1 Triticum monococcum actin (ACT-1) gene, partial cds; putative chrom

  5. Dicty_cDB: Contig-U13537-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 494_1( EF633494 |pid:none) Sitobion avenae cathepsin B-348 mR... 56 8e-10 AJ296151_1( AJ296151 |pid:none) Ostertagia...neropenaeus chinensis cathepsin... 66 2e-17 BC115254_1( BC115254 |pid:none) Danio re...obilharzia regenti cathepsin ... 53 4e-14 FN314522_1( FN314522 |pid:none) Schistosoma japonicum isolat...N357654_7( FN357654 |pid:none) Schistosoma mansoni genome sequenc... 44 2e-08 M88503_1( M88503 |pid:none) Ostertagia ostertagi cat...Contig-U13537-1Q.Seq.d (1077 letters) Database: ddbj_A 92,845,959 sequences; 95,242,211,685 total letters Searchi

  6. Enzyme-responsive doxorubicin release from dendrimer nanoparticles for anticancer drug delivery

    Directory of Open Access Journals (Sweden)

    Lee SJ

    2015-08-01

    Full Text Available Sang Joon Lee,1,* Young-Il Jeong,2,* Hyung-Kyu Park,3 Dae Hwan Kang,2,4 Jong-Suk Oh,3 Sam-Gyu Lee,5 Hyun Chul Lee31Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju, 2Biomedical Research Institute, Pusan National University Hospital, Busan, 3Department of Microbiology, Chonnam National University Medical School, Gwangju, 4Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Gyeongnam, 5Department of Physical and Rehabilitation Medicine, Chonnam National University Medical School, Gwangju, Republic of Korea*These authors contributed equally to this workBackground: Since cancer cells are normally over-expressed cathepsin B, we synthesized dendrimer-methoxy poly(ethylene glycol (MPEG-doxorubicin (DOX conjugates using a cathepsin B-cleavable peptide for anticancer drug targeting.Methods: Gly-Phe-Leu-Gly peptide was conjugated with the carboxylic acid end groups of a dendrimer, which was then conjugated with MPEG amine and doxorubicin by aid of carbodiimide chemistry (abbreviated as DendGDP. Dendrimer-MPEG-DOX conjugates without Gly-Phe-Leu-Gly peptide linkage was also synthesized for comparison (DendDP. Nanoparticles were then prepared using a dialysis procedure.Results: The synthesized DendGDP was confirmed with 1H nuclear magnetic resonance spectroscopy. The DendDP and DendGDP nanoparticles had a small particle size of less than 200 nm and had a spherical morphology. DendGDP had cathepsin B-sensitive drug release properties while DendDP did not show cathepsin B sensitivity. Further, DendGDP had improved anticancer activity when compared with doxorubicin or DendDP in an in vivo CT26 tumor xenograft model, ie, the volume of the CT26 tumor xenograft was significantly inhibited when compared with xenografts treated with doxorubicin or DendDP nanoparticles. The DendGDP nanoparticles were found to be relatively concentrated in the tumor tissue and

  7. Discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and TbcatB

    Science.gov (United States)

    Mallari, Jeremy P.; Shelat, Anang; Kosinski, Aaron; Caffrey, Conor R.; Connelly, Michele; Zhu, Fangyi; McKerrow, James H.; Guy, R. Kiplin

    2008-01-01

    Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases of T.brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention. Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured T.brucei, and specificity was assessed with a panel of four mammalian cell lines. PMID:18420405

  8. ALCALOIDES ACRIDÔNICOS INIBEM CATEPSINA L E V

    OpenAIRE

    Emerson F. Marques; Vieira, Paulo C.; Richele P. Severino

    2016-01-01

    Cathepsins represent a class of enzymes that has the primary function of randomly degrading proteins in the lysosomes, although are also involved in different pathologies. The aim of this paper was to evaluate the capacity of acridone alkaloids isolated from Swinglea glutinosa (Rutaceae) to inhibit cathepsin L in vitro . The IC50 values found were in the 0.8-57 µM range and the most promising compounds were alkaloids 1 and 2, with IC50 of 0.9 and 0.8 µM, respectively. Enzyme kinetics revealed...

  9. Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation

    Directory of Open Access Journals (Sweden)

    Swarna Oli

    2014-05-01

    Full Text Available In this paper, we report new protease inhibitory activity of plakortide E towards cathepsins and cathepsin-like parasitic proteases. We further report on its anti-parasitic activity against Trypanosoma brucei with an IC50 value of 5 μM and without cytotoxic effects against J774.1 macrophages at 100 μM concentration. Plakortide E was isolated from the sponge Plakortis halichondroides using enzyme assay-guided fractionation and identified by NMR spectroscopy and mass spectrometry. Furthermore, enzyme kinetic studies confirmed plakortide E as a non-competitive, slowly-binding, reversible inhibitor of rhodesain.

  10. Calciurn/lysosome pathway in the apoptosis of SGC-7901 cells induced by Wuxing soup%五行汤经钙离子/溶酶体途径介导的SGC-7901细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    莫非; 胡晶莹; 甘愉; 赵仰星; 朱明洁; 赵新泰; 段友容

    2011-01-01

    Objective To explore the mechanism of calcium/lysosome pathway in the apoptosis of SGC-7901 cells induced by Wuxing soup. Methods The integrity of lysosome membrane was detected by acridine orange (AO) staining,the key proteins in apoptotic pathway were tested by Western blot, and the effect of inhibitors on cell apoptotic rate and survival rate was analyzed by Annexin V-binding ELISA and CCK-8, respectively. Results The leakage of lysosome membrane was observed by AO staining. The lysosome associated apoptotic protein Cathepsin D and Cathepsin B were released into cytosol. The inhibitors of Cathepsin B, L and S exerted protection in the survival of SGC-7901 under Wuxing soup treatment in a dose-dependent manner. However, the inhibitor of Cathepsin D had no effects on apoptosis.Conclusion Wuxing soup could induce SGC-7901 cell apoptosis through calcium mediated and lysosome involved caspase-independent pathway.%目的 探索五行汤经钙离子/溶酶体途径介导的SGC-7901细胞凋亡机制.方法 吖啶橙染色检测溶酶体膜完整性,胞浆蛋白免疫印迹法检测凋亡途径蛋白变化,Annexin V结合实验和CCK-8法检测不同抑制剂对细胞凋亡和存活的影响.结果 凋亡细胞的溶酶体膜渗漏,溶酶休释放蛋白酶Cathepsin D和Cathepsin B至胞浆.天冬氨酸蛋白酶Cathepsin D抑制剂对五行汤诱导的细胞凋亡无影响;半胱氨酸蛋白酶Cathepsin B、L、S抑制荆可明显减少细胞凋亡(P<0.05),且对细胞的保护作用呈浓度依赖性.结论 五行汤经钙离子/溶酶体途径介导SGC-7901细胞凋亡.

  11. Cellular and molecular mechanisms of osteoporosis: current concepts and future direction treatment

    Directory of Open Access Journals (Sweden)

    A. T. Dolzhenko

    2016-01-01

    Full Text Available The article presents review of literature dedicated to the contemporary view on the cellular-molecular mechanisms of the bone remodeling and pathogenesis of the osteoporosis. The discovery of the cytokine RANKL-RANK-OPG system and significant role of the cathepsin K in process bone remodeling has made progress in understanding the mechanisms development disease and possible to development drugs of the new generation – denosumab, a fully human RANKL monoclonal antibody and inhibitor cathepsin K odanacatib that inhibits of the bone resorption.

  12. A selective genotyping approach identifies single nucleotide polymorphisms in porcine chromosome 2 genes associated with production and carcass traits in Italian heavy pigs

    OpenAIRE

    Vincenzo Russo; Luca Buttazzoni; Camilla Speroni; Emilio Scotti; Luca Fontanesi

    2011-01-01

    Several studies have shown that porcine chromosome 2 (SSC2) harbors important quantitative trait loci (QTL) for production traits. In particular, an imprinted QTL for muscle mass production is determined by a mutation in the IGF2 gene (intron3-g.3072G>A). We recently identified and analysed single nucleotide polymorphisms (SNPs) in genes (cathepsin D, CTSD g.70G>A; cathepsin F, CTSF g.22G>C; lactate dehydrogenase A, LDHA g.46G>T) localized on SSC2 (including the IGF2 intron3-g.3072G>A SNP) sh...

  13. Dicty_cDB: VHL488 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available ) Delia coarctata cathepsin L-like c... 159 5e-38 AF147207_1( AF147207 |pid:none) Artemia...-L mRNA,... 160 2e-38 AY363263_1( AY363263 |pid:none) Triatoma infestans cathepsin L-lik... 159 4e-38 AF194426_1( AF194426 |pid:none...g significant alignments: (bits) Value N S58669 |S58669.1 Entamoeba histolytica cysteine proteinase precursor (ACP1) gene...l cds; putative chromosome condensation factor (CCF), putative resistance protein (RGA-2), putative resistance protein...ATTTGGATCACGAAGTTTTAGCTATTGGTTATGGTACTTATCAAGGT CAAGATTATTTNTTAGNTAAAAACTC Length of 3' end seq. 506 Connected seq. ID - Connect

  14. Proteases of neutrophilic granulocytes

    OpenAIRE

    Wiesława Roszkowska-Jakimiec; Anna Worowska; Marek Gacko; Tomasz Maksimowicz

    2002-01-01

    The literature referring to proteolytic enzymes of neutrophilic granulocytes was surveyed. Biosynthesis, subcellular distribution, division according to the catalytic site structure, inhibitors and methods used to determine the activity of these enzymes were discussed. The survey included metaloproteases (granulocytic collagenase, gelatinase B), serine proteases (granulocytic elastase, cathepsin G, protease 3), membraneous proteases (aminopeptidase N, aminopeptidase P, neprilisine), cysteine ...

  15. Dicty_cDB: VFF511 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available VF (Link to library) VFF511 (Link to dictyBase) - - - Contig-U15456-1 VFF511P (Link to Original ... 57_1( DQ868657 |pid:none) Cucumis sativus aspartic proteinas ... 183 1e-80 (Q805F2) RecName: Full=Cathepsin E-B; ...

  16. Dicty_cDB: AFB825 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 53 2e-36 DQ474246_1( DQ474246 |pid:none) Lygus lineolaris cathepsin-L mRNA,... 152 3e-36 FJ349098_1( FJ349098 |pid:none) Acanthodendr...illa sp. Vietnam cathe... 152 5e-36 AF194426_1( AF194426 |pid:none) Myxine glutinos

  17. Immunohistochemical molecular markers as predictors of curability of endoscopically resected submucosal colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To clarify the usefulness of immunohistochemical molecular markers in predicting lymph node metastasis of submucosal colorectal cancer.METHODS: We examined microvessel density, lymphatic vessel density, the Ki-67 labeling index, expression of MUC1 and Matrix metalloproteinase-7 (MMP-7) in tumor cells, and expression of cathepsin D in stromal cells at the invasive front by immunostaining of samples resected from 214 patients with submucosal colorectal cancer.Pathologic features were assessed on hematoxylin-eosinstained samples. We evaluated the relations between clinicopathologic/immunohistochemical features and lymph node metastasis.RESULTS: Lesions of the superficial type, with an unfavorable histologic grade, budding, lymphatic involvement, high microvessel density (≥ 40), high lymphatic vessel density (≥ 9), high Ki-67 labeling index (≥ 42), and positivity of MUC1, cathepsin D, and MMP-7 showed a significantly high incidence of lymph node metastasis. Multivariate analysis revealed that high microvessel density, unfavorable histologic grade,cathepsin D positivity, high lymphatic vessel density,superficial type, budding, and MUC1 positivity were independent risk factors for lymph node metastasis.A combined examination with four independent immunohistochemical markers (microvessel density,cathepsin D, lymphatic vessel density, and MUC1)revealed that all lesions that were negative for all markers or positive for only one marker were negative for lymph node metastasis.CONCLUSION: Analysis of a combination of immunohistochemical molecular markers in endoscopically resected specimens of submucosal colorectal cancer allows prediction of curability regardless of the pathologic features visible of hematoxylin-eosin-stained sections.

  18. Pyknodysostose--faelles stamfar til en del af danske patienter. Udredning og molekylaergenetisk diagnostik

    DEFF Research Database (Denmark)

    Haagerup, Annette; Christensen, Mogens Fjord; Hertz, Jens Michael; Kruse, Torben A

    2002-01-01

    Eight patients with pycnodysostosis from six Danish families were examined for mutations in the cathepsin K gene. Three different mutations are the cause of pycnodysostosis in the six families--five of whom come from Ringkøbing County and one from Vejle County. One mutation has a high frequency i...

  19. Serum cysteine proteases and their inhibitors in rheumatoid arthritis: relation to disease activity and radiographic progression

    DEFF Research Database (Denmark)

    Kos, Janko; Krašovec, Marta; Troelsen, Lone;

    2011-01-01

    This study aims to investigate the serum levels of cysteine proteases cathepsins B and H and their inhibitors stefin A, stefin B, and cystatin C, as well as traditional inflammatory markers such as C-reactive protein in patients with rheumatoid arthritis and to correlate these markers with scores...

  20. Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval Western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris

    NARCIS (Netherlands)

    Bown, D.P.; Wilkinson, H.S.; Jongsma, M.A.; Gatehouse, J.A.

    2004-01-01

    Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z

  1. Expression profiles of seven channel catfish antimicrobial peptides in response to Edwardsiella ictaluri infection

    Science.gov (United States)

    Using quantitative PCR technique, the relative transcriptional levels of seven channel catfish antimicrobial peptide (AMP) genes [NK-lysin type 1, NK-lysin type 2, NK-lysin type 3, bactericidal permeability-increasing protein (BPI), cathepsin D, hepcidin, and liver-expressed antimicrobial peptide 2 ...

  2. Dietary factors impact on the association between CTSS variants and obesity related traits

    DEFF Research Database (Denmark)

    Hooton, Henri; Ängquist, Lars Henrik; Holst, Claus;

    2012-01-01

    Cathepsin S, a protein coded by the CTSS gene, is implicated in adipose tissue biology--this protein enhances adipose tissue development. Our hypothesis is that common variants in CTSS play a role in body weight regulation and in the development of obesity and that these effects are influenced by...... dietary factors--increased by high protein, glycemic index and energy diets....

  3. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase

    OpenAIRE

    Bhargava, Amol; Cotton, James A; Dixon, Brent R.; Gedamu, Lashitew; Robin M. Yates; Buret, Andre G.

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cyste...

  4. Katepsinové proteasy v patologii

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Jílková, Adéla; Mareš, Michael

    2014-01-01

    Roč. 108, č. 4 (2014), s. 358-363. ISSN 0009-2770 R&D Projects: GA ČR(CZ) GAP302/11/1481 Institutional support: RVO:61388963 Keywords : cathepsins * proteases * proteolysis Subject RIV: CE - Biochemistry Impact factor: 0.272, year: 2014

  5. A plant-insect interaction in 3D

    Czech Academy of Sciences Publication Activity Database

    Srp, Jaroslav; Pachl, Petr; Řezáčová, Pavlína; Vondrášek, Jiří; Nussbaumerová, Martina; Horn, Martin; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 55. ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 Keywords : LdCD * potato cathepsin D inhibitor * crystal structure Subject RIV: CE - Biochemistry

  6. Odanacatib, a New Drug for the Treatment of Osteoporosis: Review of the Results in Postmenopausal Women

    Directory of Open Access Journals (Sweden)

    José Luis Pérez-Castrillón

    2010-01-01

    Full Text Available Osteoclasts are specialized cells that initiate the process of bone resorption, which has two phases, dissolution of the mineral component and degradation of the organic matrix, in which cathepsin K plays a key role. Cathepsin K inhibitors, which block the activity of cathepsin on bone resorption lacunae, may be a new therapeutic option in osteoporosis. Odanacatib is a nonpeptidic biaryl inhibitor of cathepsin K. Two studies have evaluated the efficacy and safety of odanacatib, a phase I study to determine the dose and a phase II study of safety and efficacy. Due to the long half-life of odanacatib and the similar effects of different doses on bone remodeling markers, a weekly dosage was chosen for the phase II trail, with the best results being obtained with a dose of 50 mg. At 36 months, increases in bone mineral density similar to those produced by other powerful antiresorptive drugs (zoledronate and denosumab were observed but there were differences in the behaviour of bone remodeling markers. Data on fractures from the phase III trial currently in development are required to confirm these possible advantages.

  7. Simukunin from the Salivary Glands of the Black Fly Simulium vittatum Inhibits Enzymes That Regulate Clotting and Inflammatory Responses

    Czech Academy of Sciences Publication Activity Database

    Tsujimoto, H.; Kotsyfakis, Michalis; Francischetti, I.M.B.; Eum, J. H.; Strand, M. R.; Champagne, D. E.

    2012-01-01

    Roč. 7, č. 2 (2012), e29964. E-ISSN 1932-6203 Institutional research plan: CEZ:AV0Z60220518 Keywords : Simulium vittatum * Kunitz-family protein * elastase * cathepsin G * Factor Xa * coagulation * inflammation * serine proteases * BPTI-like inhibitor * Simukunin Subject RIV: EC - Immunology Impact factor: 3.730, year: 2012

  8. Effects of Odanacatib on the Radius and Tibia of Postmenopausal Women

    DEFF Research Database (Denmark)

    Cheung, Am; Majumdar, S; Brixen, K;

    2014-01-01

    The cathepsin K inhibitor odanacatib, currently in phase 3 development for postmenopausal osteoporosis, has a novel mechanism of action that reduces bone resorption while maintaining bone formation. In phase 2 studies, odanacatib increased areal BMD at the lumbar spine and total hip progressively...

  9. A cluster of hematopoietic serine protease genes is found on the same chromosomal band as the human α/δ T-cell receptor locus

    International Nuclear Information System (INIS)

    The chymotrypsin-like family of serine protease genes includes several members that are expressed exclusively in subsets of hematopoietic cells. For example, human neutrophil elastase and cathepsin G are expressed only in myelomonocytic precursors, and cytotoxic-T-cell serine proteases are found only in cytotoxic lymphocytes. The authors have used a cathepsin G cDNA probe to clone two cathepsin G-like genes (designated CGL-1 and CGL-2) from a human genomic library. They have determined that CGL-1 is identical to a previously identified gene (known as CCPI, CTLA I, or cytotoxic serine protease B) that is expressed only in activated cytotoxic T lymphocytes. They show here that cathepsin G, CGL-1, and CGL-2 are linked on an ∼50-kilobase locus found on human chromosome 14 at band q11.2. This gene cluster maps to the same chromosomal band as the α and δ T-cell receptor genes; this region is involved in most chromosomal translocations and inversions that are specifically associated with T-cell malignancies

  10. Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors.

    Science.gov (United States)

    O'Donoghue, Anthony J; Ivry, Sam L; Chaudhury, Chaity; Hostetter, Daniel R; Hanahan, Douglas; Craik, Charles S

    2016-09-01

    The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection. PMID:27149201

  11. Hypoxia enhances the antiglioma cytotoxicity of B10, a glycosylated derivative of betulinic acid.

    Science.gov (United States)

    Fischer, Sebastian; Ronellenfitsch, Michael W; Thiepold, Anna-Luisa; Harter, Patrick N; Reichert, Sebastian; Kögel, Donat; Paschke, Reinhard; Mittelbronn, Michel; Weller, Michael; Steinbach, Joachim P; Fulda, Simone; Bähr, Oliver

    2014-01-01

    B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma. PMID:24743710

  12. Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II

    DEFF Research Database (Denmark)

    Henriksen, Kim; Gram, Jeppe; Schaller, Sophie;

    2004-01-01

    , the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the...

  13. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C;

    2004-01-01

    Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...

  14. Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger.

    Science.gov (United States)

    Markosyan, Ruben M; Miao, Chunhui; Zheng, Yi-Min; Melikyan, Gregory B; Liu, Shan-Lu; Cohen, Fredric S

    2016-01-01

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry. PMID:26730950

  15. The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

    Science.gov (United States)

    Bailleul, Geoffrey; Kravtzoff, Amanda; Joulin-Giet, Alix; Lecaille, Fabien; Labas, Valérie; Meudal, Hervé; Loth, Karine; Teixeira-Gomes, Ana-Paula; Gilbert, Florence B; Coquet, Laurent; Jouenne, Thierry; Brömme, Dieter; Schouler, Catherine; Landon, Céline; Lalmanach, Gilles; Lalmanach, Anne-Christine

    2016-01-01

    Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion. PMID:27561012

  16. Huntingtin cleavage product A forms in neurons and is reduced by gamma-secretase inhibitors

    Directory of Open Access Journals (Sweden)

    Betschart Claudia

    2010-12-01

    Full Text Available Abstract Background The mutation in Huntington's disease is a polyglutamine expansion near the N-terminus of huntingtin. Huntingtin expressed in immortalized neurons is cleaved near the N-terminus to form N-terminal polypeptides known as cleavage products A and B (cpA and cpB. CpA and cpB with polyglutamine expansion form inclusions in the nucleus and cytoplasm, respectively. The formation of cpA and cpB in primary neurons has not been established and the proteases involved in the formation of these fragments are unknown. Results Delivery of htt cDNA into the mouse striatum using adeno-associated virus or into primary cortical neurons using lentivirus generated cpA and cpB, indicating that neurons in brain and in vitro can form these fragments. A screen of small molecule protease inhibitors introduced to clonal striatal X57 cells and HeLa cells identified compounds that reduced levels of cpA and are inhibitors of the aspartyl proteases cathepsin D and cathepsin E. The most effective compound, P1-N031, is a transition state mimetic for aspartyl proteases. By western blot analysis, cathepsin D was easily detected in clonal striatal X57 cells, mouse brain and primary neurons, whereas cathepsin E was only detectible in clonal striatal X57 cells. In primary neurons, levels of cleavage product A were not changed by the same compounds that were effective in clonal striatal cells or by mRNA silencing to partially reduce levels of cathepsin D. Instead, treating primary neurons with compounds that are known to inhibit gamma secretase activity either indirectly (Imatinib mesylate, Gleevec or selectively (LY-411,575 or DAPT reduced levels of cpA. LY-411,575 or DAPT also increased survival of primary neurons expressing endogenous full-length mutant huntingtin. Conclusion We show that cpA and cpB are produced from a larger huntingtin fragment in vivo in mouse brain and in primary neuron cultures. The aspartyl protease involved in forming cpA has cathepsin

  17. Potential role of odanacatib in the treatment of osteoporosis

    Directory of Open Access Journals (Sweden)

    Ng KW

    2012-07-01

    Full Text Available Kong Wah NgDepartment of Endocrinology and Diabetes and St Vincent’s Institute, St Vincent’s Hospital, Fitzroy, Victoria, AustraliaAbstract: Cathepsin K is a key enzyme involved in the degradation of organic bone matrix by osteoclasts. Inhibition of bone resorption observed in human and animal models deficient for cathepsin K has identified this enzyme as a suitable target for intervention by small molecules with the potential to be used as therapeutic agents in the treatment of osteoporosis. Odanacatib (ODN is a nonbasic selective cathepsin K inhibitor with good pharmacokinetic parameters such as minimal in vitro metabolism, long half-life, and oral bioavailability. In preclinical studies, ovariectomized monkeys and rabbits treated with ODN showed substantial inhibition of bone resorption markers along with increases in bone mineral density (BMD. Significant differences were observed in the effects of ODN treatment compared with those of other antiresorptive agents such as bisphosphonates and denosumab. ODN displayed compartment-specific effects on trabecular versus cortical bone formation, with treatment resulting in marked increases in periosteal bone formation and cortical thickness in ovariectomized monkeys whereas trabecular bone formation was reduced. Furthermore, osteoclasts remained viable. Phase I and II studies conducted in postmenopausal women showed ODN to be safe and well tolerated. After 5 years, women who received ODN 50 mg weekly continuously from year 1 (n = 13, showed BMD increases from baseline of 11.9% at the lumbar spine, 9.8% at the femoral neck, 10.9% at the hip trochanter, and 8.5% at the total hip. Additionally, these subjects maintained a low level of the urine bone resorption marker N-terminal telopeptide/creatinine (-67.4% from baseline through 5 years of treatment, while levels of serum bone-specific alkaline phosphatase remained only slightly reduced relative to baseline (-15.3%. In women who were switched from

  18. Dicty_cDB: SSH357 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available k Frame C: ---wcstwssiccn*cfp**fptlyiwylf*akmfpn*igswcfscwlwcsr*xc*rssi nrkpthcypqd*gy*s*ilc*fic*cwpkgn*lldr...cysteine proteinase 2. 603 e-169 1 M16039 |M16039.1 Dictyostelium discoideum pst-cath gene encoding pst-cath...1_2_D09.b1_A006 G5 trophont cDNA (INIT1) Ichthyophthirius multifiliis cDNA, mRNA ... Dictyostelium discoideum cysteine prot... 93 4e-18 AF412313_1( AF412313 |pid:none) Haemonchus contortus cath...epsin L c... 89 5e-17 AY004155_1( AY004155 |pid:none) Haemonchus contortus cathepsin L c... 89 5e-17 BC0758

  19. Dicty_cDB: Contig-U08336-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e) Nicotiana benthamiana cysteine pro... 33 4.1 AJ319727_1( AJ319727 |pid:none) Leishmania mexicana...e: Full=Testin-2; Contains: RecName: Full=Testi... 32 5.3 AK145933_1( AK145933 |pid:none) Mus musculus 13 days pregnant... L-like proteinase; EC=... 42 0.007 EF611824_1( EF611824 |pid:none) Fascio...e) Fasciola gigantica cathepsin L (ca... 42 0.009 AY950578_1( AY950578 |pid:none) Paralichthys olivac...e Porto 1 ca... 46 5e-04 AF239265_1( AF239265 |pid:none) Fasciola gigantica cathepsin

  20. Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

    DEFF Research Database (Denmark)

    Nylandsted, J; Jäättelä, M; Hoffmann, E K;

    2004-01-01

    Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found......) and Na(+),K(+),2Cl(-)-cotransporter (NKCC1) to RVI. Hypertonic stress induced caspase-3 activity in WEHI cells and iMEFs, an effect potentiated by Hsp70 in WEHI cells but inhibited by Hsp70 in iMEFs. Osmotic shrinkage-induced PCD was associated with Hsp70-inhibitable cysteine cathepsin release in i......MEFs and attenuated by caspase and cathepsin inhibitors in WEHI cells. Treatment with TNF-alpha or the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) reduced the viability of WEHI cells further under isotonic and mildly, but not severely, hypertonic conditions. Thus, it is concluded that shrinkage...