Identification of novel serine proteinase gene transcripts in the midguts of two tropical insect pests, Scirpophaga incertulas (Wk.) and Helicoverpa armigera (Hb.).

PubMed

Mazumdar-Leighton, S; Babu, C R; Bennett, J

2000-01-01

We have used RT PCR and 3'RACE to identify diverse serine proteinase genes expressed in the midguts of the rice yellow stem borer (Scirpophaga incertulas) and Asian corn borer (Helicoverpa armigera). The RT-PCR primers encoded the conserved regions around the active site histidine57 and serine195 of Drosophila melanogaster alpha trypsin, including aspartate189 of the specificity pocket. These primers amplified three transcripts (SiP1-3) from midguts of S. incertulas, and two transcripts (HaP1-2) from midguts of H. armigera. The five RT PCR products were sequenced to permit design of gene-specific forward primers for use with anchored oligo dT primers in 3'RACE. Sequencing of the 3'RACE products indicated that SiP1, SiP2 and HaP1 encoded trypsin-like serine proteinases, while HaP2 encoded a chymotrypsin-like serine proteinases. The SiP3 transcript proved to be an abundant 960 nt mRNA encoding a trypsin-like protein in which the active site serine195 was replaced by aspartate. The possible functions of this unusual protein are discussed.

Frequency of Tabagism and N34S and P55S Mutations of Serine Peptidase Inhibitor, Kazal Type 1 (SPINK1) and R254W Mutation of Chymotrypsin C (CTRC) in Patients With Chronic Pancreatitis and Controls.

PubMed

da Costa, Marianges Zadrozny Gouvêa; Pires, Júlia Glória Lucatelli; Nasser, Paulo Dominguez; Ferreira, Camila da Silva; Teixeira, Ana Cristina de Sá; Paranaguá-Vezozzo, Denise Cerqueira; Guarita, Dulce Reis; Carrilho, Flair José; Ono, Suzane Kioko

2016-10-01

This study aimed to investigate the association between chronic pancreatitis and smoking or genetic mutations. The study sample comprised 148 patients with chronic pancreatitis, 110 chronic alcoholic subjects without pancreatic disease, and 297 volunteer blood donors. Of the patients with chronic pancreatitis, 74% had alcoholic etiology and 26% had idiopathic pancreatitis. The frequency of smoking was 91.4% in patients with alcoholic pancreatitis, higher than 73.3% in alcoholic subjects without pancreatitis (P < 0.01). The difference in smoking frequency was not significant between the patients with idiopathic pancreatitis and blood donors. The N34S mutation of serine peptidase inhibitor, Kazal type 1 (SPINK1) was found in 2.7% of patients with chronic alcoholic pancreatitis, in 5.3% of patients with idiopathic pancreatitis, and in 0.4% of blood donors (P = 0.02). The P55S mutation of SPINK1 was found in 2.7% of patients with alcoholic pancreatitis and in 0.7% of blood donors (P = 0.12). The R254W mutation of chymotrypsin C was found in 0.9% of patients with alcoholic pancreatitis, in 0.9% of chronic alcoholic subjects without pancreatitis, and in 0.4% of blood donors (P = 0.75). In all cases, the mutations were heterozygous. Smoking and the N34S mutation of SPINK1 were positively correlated with chronic pancreatitis.

Serine Protease Inhibitor Kazal Type 1 (SPINK1) c.194+2T > C Mutation May Predict Long-term Outcome of Endoscopic Treatments in Idiopathic Chronic Pancreatitis.

PubMed

Sun, Chang; Liu, Mu-Yun; Liu, Xiao-Gang; Hu, Liang-Hao; Xia, Tian; Liao, Zhuan; Li, Zhao-Shen

2015-11-01

Endoscopic interventional is a commonly used treatment method for idiopathic chronic pancreatitis. Serine protease inhibitor Kazal type 1 (SPINK1) 194+2T>C mutation is most frequently observed in Chinese pancreatitis patients and influences the clinical course of idiopathic chronic pancreatitis patients. We conducted this study to determine the impacts of this mutation on the outcome of endoscopic treatments.In this study, we enrolled 423 patients. Among them, 101 idiopathic chronic pancreatitis patients without other relevant mutations had a successful endoscopic procedure and completed follow-up. Clinical characteristics including Izbicki pain score, exocrine and endocrine function, were evaluated. Genetic sequencing was conducted to detect SPINK1 194+2T>C mutations.The c.194+2T>C mutation was found in 58 (57.43%) patients. Factors relevant to pain relief are c.194+2T>C mutation (P = 0.011), severe pain before treatment (P = 0.005), and necessary subsequent endoscopic treatments (P < 0.001). More patients with the intronic mutation had deteriorated endocrine function (P = 0.001) relative to those patients without the mutation.Patients carrying the c.194+2T>C mutation were less likely to achieve pain relief through endoscopic treatments. They also have a higher risk of endocrine function deterioration. SPINK1 c.194+2T>C mutation may be applied as a pretreatment predictor in idiopathic chronic pancreatitis patients.

Cloned cytolytic T-effector cells and their malignant variants produce an extracellular matrix degrading trypsin-like serine proteinase.

PubMed Central

Simon, M M; Simon, H G; Fruth, U; Epplen, J; Müller-Hermelink, H K; Kramer, M D

1987-01-01

This report describes the distribution of a trypsin-like proteinase in defined homogeneous cytolytic T-cell lines (CTLL) and their in vitro and in vivo derived malignant T-lymphoma variants. By means of chromogenic peptide substrates, we found the enzyme to attack preferentially at the carboxy terminus of arginine, in particular when non-polar amino acids were present in the amino terminal neighbouring position. The enzyme was identified by means of various inhibitors as a serine type proteinase having a pH optimum around 8 X 5. Affinity chromatography in connection with molecular sieving resulted in a 200-fold purification and indicated a molecular weight (MW) of about 50,000 for the proteinase. The enzyme was found to be highly expressed in antigen-specific CTLL as well as in their tumorigenic variants. Both intact lymphocytes of all CTLL tested and Triton X-100 lysates or enriched proteinase preparations thereof were able to degrade a high molecular weight protein (casein) and to release high molecular weight split products from the sulphated proteoglycans in subendothelial extracellular matrix. The results are discussed with respect to the invasiveness of normal and malignant T lymphocytes and the proteinase is suggested to be crucially involved in the process of cellular migration in vivo. Images Figure 1 PMID:3546101

Epoxy-α-Lapachone Has In Vitro and In Vivo Anti-Leishmania (Leishmania) amazonensis Effects and Inhibits Serine Proteinase Activity in This Parasite

PubMed Central

Souza-Silva, Franklin; Bourguignon, Saulo Cabral; Pereira, Bernardo Acácio Santini; Côrtes, Luzia Monteiro de Castro; de Oliveira, Luiz Filipe Gonçalves; Henriques-Pons, Andrea; Finkelstein, Lea Cysne; Ferreira, Vitor Francisco; Carneiro, Paula Fernandes; de Pinho, Rosa Teixeira; Caffarena, Ernesto Raul

2015-01-01

Leishmania (Leishmania) amazonensis is a protozoan that causes infections with a broad spectrum of clinical manifestations. The currently available chemotherapeutic treatments present many problems, such as several adverse side effects and the development of resistant strains. Natural compounds have been investigated as potential antileishmanial agents, and the effects of epoxy-α-lapachone on L. (L.) amazonensis were analyzed in the present study. This compound was able to cause measurable effects on promastigote and amastigote forms of the parasite, affecting plasma membrane organization and leading to death after 3 h of exposure. This compound also had an effect in experimentally infected BALB/c mice, causing reductions in paw lesions 6 weeks after treatment with 0.44 mM epoxy-α-lapachone (mean lesion area, 24.9 ± 2.0 mm2), compared to untreated animals (mean lesion area, 30.8 ± 2.6 mm2) or animals treated with Glucantime (mean lesion area, 28.3 ± 1.5 mm2). In addition, the effects of this compound on the serine proteinase activities of the parasite were evaluated. Serine proteinase-enriched fractions were extracted from both promastigotes and amastigotes and were shown to act on specific serine proteinase substrates and to be sensitive to classic serine proteinase inhibitors (phenylmethylsulfonyl fluoride, aprotinin, and antipain). These fractions were also affected by epoxy-α-lapachone. Furthermore, in silico simulations indicated that epoxy-α-lapachone can bind to oligopeptidase B (OPB) of L. (L.) amazonensis, a serine proteinase, in a manner similar to that of antipain, interacting with an S1 binding site. This evidence suggests that OPB may be a potential target for epoxy-α-lapachone and, as such, may be related to the compound's effects on the parasite. PMID:25583728

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

PubMed Central

Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

1997-01-01

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

Matrix metalloproteinase and heparin-stimulated serine proteinase activities in post-prostate massage urine of men with prostate cancer.

PubMed

Muñoz, David; Serrano, Maria K; Hernandez, Maria E; Haller, Ross; Swanson, Tamara; Slaton, Joel W; Sinha, Akhouri A; Wilson, Michael J

2017-12-01

Proteinases secreted by the prostate gland have a reproductive function in cleaving proteins in the ejaculate and in the female reproductive tract, but some may have a fundamental role in disease and pathological processes including cancer. The purpose of this study was to determine if there were differences in proteinase activities in urine samples collected following prostate massage of men positive (CaP) or negative (no evidence of malignancy, NEM) for biopsy determined prostate cancer. Matrix metalloproteinase (MMP) and serine proteinase activities were detected using protein substrate zymography. There were no differences in activities of MMP-2, proMMP-9, and MMP-9/NGAL (neutrophil gelatinase associated lipocalin) complex (gelatin substrate) in men with detected prostate cancer, although the latter two were somewhat diminished. A caseinolytic activity of about 75kDa inhibited by calcium did not differ between the NEM and CaP groups. Heparin stimulated calcium sensitive gelatinolytic activities of approximately 22, 42, and 60kDa, but did not affect activities of MMP-2, MMP-9, or the 75kDa caseinolytic activity. The 22, 42, and 60kDa activities appear to be serine proteinases since they were inhibited by benzamidine. There was a significant decrease in the 22kDa heparin-stimulated serine proteinase activity in urines of men with cancer. Proteinase expression and activities, perhaps in combination with other potential markers, may prove useful in urine for detection and evaluation of prostate cancer. Copyright © 2017. Published by Elsevier Inc.

A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.

PubMed

Oliveira-Neto, Osmundo B; Batista, João A N; Rigden, Daniel J; Fragoso, Rodrigo R; Silva, Rodrigo O; Gomes, Eliane A; Franco, Octávio L; Dias, Simoni C; Cordeiro, Célia M T; Monnerat, Rose G; Grossi-De-Sá, Maria F

2004-09-01

Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated. Copyright 2004 Elsevier Ltd.

Proteinase-Activated Receptor 2 May Drive Cancer Progression by Facilitating TGF-β Signaling.

PubMed

Ungefroren, Hendrik; Witte, David; Rauch, Bernhard H; Settmacher, Utz; Lehnert, Hendrik; Gieseler, Frank; Kaufmann, Roland

2017-11-22

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β (TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-β signaling-promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection.

Proteinase-Activated Receptor 2 May Drive Cancer Progression by Facilitating TGF-β Signaling

PubMed Central

Ungefroren, Hendrik; Witte, David; Settmacher, Utz; Lehnert, Hendrik; Kaufmann, Roland

2017-01-01

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β (TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-β signaling–promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection. PMID:29165389

Identification and activity of a lower eukaryotic serine proteinase inhibitor (serpin) from Cyanea capillata: analysis of a jellyfish serpin, jellypin.

PubMed

Cole, Elisabeth B; Miller, David; Rometo, David; Greenberg, Robert M; Brömme, Dieter; Cataltepe, Sule; Pak, Stephen C; Mills, David R; Silverman, Gary A; Luke, Cliff J

2004-09-21

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved approximately 1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, approximately 1000 MYA.

Influence of white spot syndrome virus infection on hepatopancreas gene expression of `Huanghai No. 2' shrimp ( Fenneropenaeus chinensis)

NASA Astrophysics Data System (ADS)

Meng, Xianhong; Shi, Xiaoli; Kong, Jie; Luan, Sheng; Luo, Kun; Cao, Baoxiang; Liu, Ning; Lu, Xia; Li, Xupeng; Deng, Kangyu; Cao, Jiawang; Zhang, Yingxue; Zhang, Hengheng

2017-10-01

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of `Huanghai No. 2' ( Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48-96 h), peak (168-192 h) and late (264-288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.

PepJ is a new extracellular proteinase of Aspergillus nidulans.

PubMed

Emri, T; Szilágyi, M; László, K; M-Hamvas, M; Pócsi, I

2009-01-01

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 degrees C, respectively. The enzyme was stable at pH 3.5-10.5 and was still active at 95 degrees C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.

Assessment of nematode resistance in wheat transgenic plants expressing potato proteinase inhibitor (PIN2) gene.

PubMed

Vishnudasan, Dalia; Tripathi, M N; Rao, Uma; Khurana, Paramjit

2005-10-01

Serine proteinase inhibitors (IP's) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T(0) transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. chi(2) analysis reveals the stable integration and segregation of the genes in both the T(1) and T(2) progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number.

Chemically modified tetracycline-3 (CMT-3): a novel inhibitor of the serine proteinase, elastase.

PubMed

Gu, Ying; Lee, Hsi-Ming; Simon, Sanford R; Golub, Lorne M

2011-12-01

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α(1)-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: comparison of wild and in vitro propagated plants.

PubMed

Patiño, Arley Camilo; Benjumea, Dora María; Pereañez, Jaime Andrés

2013-09-16

The plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. We have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. Colorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. Our results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases. Crown Copyright © 2013 Published by Elsevier Ireland Ltd. All rights reserved.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

PubMed Central

Nagaike, Koki; Kawaguchi, Makiko; Takeda, Naoki; Fukushima, Tsuyoshi; Sawaguchi, Akira; Kohama, Kazuyo; Setoyama, Mitsuru; Kataoka, Hiroaki

2008-01-01

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1+/+ blastocysts with Hai-1/Spint1−/− embryonic stem cells successfully generated high-chimeric Hai-1/Spint1−/− embryos (B6Hai-1−/−High) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1−/− mice was caused by placental dysfunction. However, newborn B6Hai-1−/−High mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development. PMID:18832587

Molecular cloning, expression and characterization of a serine proteinase inhibitor gene from Entamoeba histolytica.

PubMed

Riahi, Yael; Siman-Tov, Rama; Ankri, Serge

2004-02-01

Serine proteinase inhibitors (serpins) are irreversible suicide inhibitors of proteinases that regulate a wide range of biological processes, including pathogen evasion of the host defence system. We report the cloning and characterization of a gene encoding a serpin from the protozoan parasite Entamoeba histolytica (Ehserp) that may function in this manner. The protein encoded by Ehserp contains 371 amino acids with a predicted mass of 42.6 kDa. Antibodies to a 42 kDa recombinant Ehserp react specifically with two bands of 42 and 49 kDa in trophozoite extracts. Ehserp has a cytoplasmic localization and is secreted by trophozoites incubated in the presence of mammalian cells, but not by resting trophozoites. A panel of mammalian serine proteinases was screened, but none of them was inhibited by the recombinant Ehserp. In contrast, the 49 kDa Ehserp present in the secretion product (SP) of activated macrophages interacted with human neutrophil cathepsin G to form a complex resistant to sodium dodecyl sulphate. We discuss the nature of the 42 and 49 kDa Ehserp and the possible roles that Ehserp may play in the survival of the parasite inside the host.

Evolutionary patterns of proteinase activity in attine ant fungus gardens

PubMed Central

2011-01-01

Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes. PMID:21247468

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis.

PubMed

Lakhal, Samira; Schödel, Johannes; Townsend, Alain R M; Pugh, Christopher W; Ratcliffe, Peter J; Mole, David R

2011-02-11

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.

Streptococcus thermophilus Cell Wall-Anchored Proteinase: Release, Purification, and Biochemical and Genetic Characterization

PubMed Central

Fernandez-Espla, María Dolores; Garault, Peggy; Monnet, Véronique; Rul, Françoise

2000-01-01

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca2+ ions. Its activity was optimal at 37°C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria. PMID:11055922

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

PubMed

Zhang, Daimeng; Wan, Weisong; Kong, Tongtong; Zhang, Ming; Aweya, Jude Juventus; Gong, Yi; Li, Shengkang

2018-05-07

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His 156 , Asp 226 , Ser 321 ). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain. Copyright © 2018. Published by Elsevier Ltd.

Identification of a female spawn-associated Kazal-type inhibitor from the tropical abalone Haliotis asinina.

PubMed

Wang, Tianfang; Nuurai, Parinyaporn; McDougall, Carmel; York, Patrick S; Bose, Utpal; Degnan, Bernard M; Cummins, Scott F

2016-07-01

Abalone (Haliotis) undergoes a period of reproductive maturation, followed by the synchronous release of gametes, called broadcast spawning. Field and laboratory studies have shown that the tropical species Haliotis asinina undergoes a two-week spawning cycle, thus providing an excellent opportunity to investigate the presence of endogenous spawning-associated peptides. In female H. asinina, we have isolated a peptide (5145 Da) whose relative abundance in hemolymph increases substantially just prior to spawning and is still detected using reverse-phase high-performance liquid chromatography chromatograms up to 1-day post-spawn. We have isolated this peptide from female hemolymph as well as samples prepared from the gravid female gonad, and demonstrated through comparative sequence analysis that it contains features characteristic of Kazal-type proteinase inhibitors (KPIs). Has-KPI is expressed specifically within the gonad of adult females. A recombinant Has-KPI was generated using a yeast expression system. The recombinant Has-KPI does not induce premature spawning of female H. asinina when administered intramuscularly. However it displays homomeric aggregations and interaction with at least one mollusc-type neuropeptide (LRDFVamide), suggesting a role for it in regulating neuropeptide endocrine communication. This research provides new understanding of a peptide that can regulate reproductive processes in female abalone, which has the potential to lead to the development of greater control over abalone spawning. The findings also highlight the need to further explore abalone reproduction to clearly define a role for novel spawning-associated peptide in sexual maturation and spawning. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus).

PubMed

Liu, Zi-Qiang; Tuo, Feng-Yan; Song, Liang; Liu, Yu-Xin; Dong, Xiu-Ping; Li, Dong-Mei; Zhou, Da-Yong; Shahidi, Fereidoon

2018-08-01

Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter. Copyright © 2018 Elsevier Ltd. All rights reserved.

Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors.

PubMed

Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P

2014-05-01

Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s). Copyright © 2014 Elsevier Ltd. All rights reserved.

Rare hereditary cause of chronic pancreatitis in a young male: SPINK1 mutation

PubMed Central

Patel, Janaki; Madan, Arina; Gammon, Amanda; Sossenheimer, Michael; Samadder, Niloy Jewel

2017-01-01

Hereditary chronic pancreatitis associated with a mutation in the serine protease inhibitor, Kazal Type-1 (SPINK-1 gene) is extremely rare. The SPINK1 mutation results in trypsinogen activation which predisposes to chronic pancreatitis predominately when combined with CFTR gene mutations. It presents as either chronic or recurrent acute pancreatitis. Symptom control and management of complications is important. Active surveillance with cross-sectional imaging for pancreatic malignancy in individuals with hereditary pancreatitis is advocated due to individuals being high risk. We present an unusual case of a young male who initially presented with renal colic and was incidentally diagnosed with severe chronic pancreatitis on abdominal imaging, with genetic testing confirming a homozygous SPINK1 mutation. PMID:29515728

Rare hereditary cause of chronic pancreatitis in a young male: SPINK1 mutation.

PubMed

Patel, Janaki; Madan, Arina; Gammon, Amanda; Sossenheimer, Michael; Samadder, Niloy Jewel

2017-01-01

Hereditary chronic pancreatitis associated with a mutation in the serine protease inhibitor, Kazal Type-1 (SPINK-1 gene) is extremely rare. The SPINK1 mutation results in trypsinogen activation which predisposes to chronic pancreatitis predominately when combined with CFTR gene mutations. It presents as either chronic or recurrent acute pancreatitis. Symptom control and management of complications is important. Active surveillance with cross-sectional imaging for pancreatic malignancy in individuals with hereditary pancreatitis is advocated due to individuals being high risk. We present an unusual case of a young male who initially presented with renal colic and was incidentally diagnosed with severe chronic pancreatitis on abdominal imaging, with genetic testing confirming a homozygous SPINK1 mutation.

Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against α-Amylase, Serine Proteinases and Fungi.

PubMed

Vieira Bard, Gabriela C; Nascimento, Viviane V; Ribeiro, Suzanna F F; Rodrigues, Rosana; Perales, Jonas; Teixeira-Ferreira, André; Carvalho, André O; Fernandes, Katia Valevski S; Gomes, Valdirene M

2015-04-01

Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of α-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus α-amylases as well as serine proteinases.

A serine proteinase homologue, SPH-3, plays a central role in insect immunity.

PubMed

Felföldi, Gabriella; Eleftherianos, Ioannis; Ffrench-Constant, Richard H; Venekei, István

2011-04-15

Numerous vertebrate and invertebrate genes encode serine proteinase homologues (SPHs) similar to members of the serine proteinase family, but lacking one or more residues of the catalytic triad. These SPH proteins are thought to play a role in immunity, but their precise functions are poorly understood. In this study, we show that SPH-3 (an insect non-clip domain-containing SPH) is of central importance in the immune response of a model lepidopteran, Manduca sexta. We examine M. sexta infection with a virulent, insect-specific, Gram-negative bacterium Photorhabdus luminescens. RNA interference suppression of bacteria-induced SPH-3 synthesis severely compromises the insect's ability to defend itself against infection by preventing the transcription of multiple antimicrobial effector genes, but, surprisingly, not the transcription of immune recognition genes. Upregulation of the gene encoding prophenoloxidase and the activity of the phenoloxidase enzyme are among the antimicrobial responses that are severely attenuated on SPH-3 knockdown. These findings suggest the existence of two largely independent signaling pathways controlling immune recognition by the fat body, one governing effector gene transcription, and the other regulating genes encoding pattern recognition proteins.

Complementary effect of hydroquinone and retinoic acid on corneocyte desquamation with their combination use.

PubMed

Cheong, Kyung Ah; Lee, Tae Ryong; Lee, Ai-Young

2017-08-01

Retinoic acid (RA) enhances skin-lightening capabilities of hydroquinone (HQ), at least in part, by facilitating desquamation which leads to increase penetration of HQ. The desquamation also affects skin irritation levels. The mechanism of RA-induced desquamation, however, has not been completely explored and no such data has been available for HQ uses. To examine the role of HQ, RA, and their combination in the desquamation. Primary cultured normal human keratinocytes, which were treated with HQ and/or RA in presence or absence of serine-specific inhibitor Kazal type5 (SPINK5)/lympho-epithelial Kazal-type-related inhibitor (LEKTI) knockdown or recombinant human SPINK5/LEKTI, and biopsied skin samples applied with HQ or RA were examined. Expression levels of corneodesmosin (CDSN), desmocollin1 (DSC1), kallikrein5 (KLK5), KLK7, and SPINK5/LEKTI, and proteolysis activity against extracted human skin epidermal protein were determined using time-course real-time PCR, Western blotting, ELISA, and immunofluorescence staining. HQ increased but RA decreased the synthesis of CDSN and DSC1. HQ reduced corneodesmosome degradation by the upregulation of SPINK5/LEKTI, whereas RA showed opposite results without upregulation of SPINK5/LEKTI. The combination of HQ and RA was close to the sum of the individual components. HQ reduced corneocyte desquamation. However, RA enhanced desquamation. The combination induced more desquamation than HQ but less than RA. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

USDA-ARS?s Scientific Manuscript database

Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

Specificity of the collagenolytic serine proteinase from the pancreas of the catfish (Parasilurus asotus).

PubMed

Yoshinaka, R; Sato, M; Yamashita, M; Itoko, M; Ikeda, S

1987-01-01

The collagenolytic serine proteinase from the pancreas of the catfish (Parasilus asotus) had a pH optimum of 7.5 for native, reconstituted calf skin collagen fibrils. The enzyme was most stable at pH 6-9. The enzyme hydrolyzed heat-denatured collagen (gelatin), casein, hemoglobin and elastin in addition to native collagen but not virtually Tos-Arg-OEe, Bz-Tyr-OEe and Suc-(Ala)3-NA. The enzyme cleaved Leu-Gly (or Gln-Gly), Gly-Ile and Ile-Ala bonds on DNP-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH2 and DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg.

Alterations in the Helicoverpa armigera Midgut Digestive Physiology after Ingestion of Pigeon Pea Inducible Leucine Aminopeptidase

PubMed Central

Lomate, Purushottam R.; Jadhav, Bhakti R.; Giri, Ashok P.; Hivrale, Vandana K.

2013-01-01

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory. PMID:24098675

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

PubMed

Lomate, Purushottam R; Jadhav, Bhakti R; Giri, Ashok P; Hivrale, Vandana K

2013-01-01

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

PubMed Central

Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

1990-01-01

The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2312150

Functional analysis of Agaricus bisporus serine proteinase 1 reveals roles in utilization of humic rich substrates and adaptation to the leaf‐litter ecological niche

PubMed Central

Heneghan, Mary N.; Burns, Claire; Costa, Ana M. S. B.; Burton, Kerry S.; Challen, Michael P.

2016-01-01

Summary Agaricus bisporus is a secondary decomposer fungus and an excellent model for the adaptation, persistence and growth of fungi in humic‐rich environments such as soils of temperate woodland and pastures. The A. bisporus serine proteinase SPR1 is induced by humic acids and is highly expressed during growth on compost. Three Spr1 gene silencing cassettes were constructed around sense, antisense and non‐translatable‐stop strategies (pGRsensehph, pGRantihph and pGRstophph). Transformation of A. bisporus with these cassettes generated cultures showing a reduction in extracellular proteinase activity as demonstrated by the reduction, or abolition, of a clearing zone on plate‐based bioassays. These lines were then assessed by detailed enzyme assay, RT‐qPCR and fruiting. Serine proteinase activity in liquid cultures was reduced in 83% of transformants. RT‐qPCR showed reduced Spr1 mRNA levels in all transformants analysed, and these correlated with reduced enzyme activity. When fruiting was induced, highly‐silenced transformant AS5 failed to colonize the compost, whilst for those that did colonize the compost, 60% gave a reduction in mushroom yield. Transcriptional, biochemical and developmental observations, demonstrate that SPR1 has an important role in nutrient acquisition in compost and that SPR1 is a key enzyme in the adaptation of Agaricus to the humic‐rich ecological niche formed during biomass degradation. PMID:27113919

Insect resistance to sugar beet pests mediated by a Beta vulgaris proteinase inhibitor transgene

USDA-ARS?s Scientific Manuscript database

We transformed sugar beet (Beta vulgaris) hairy roots and Nicotiana benthamiana plants with a Beta vulgaris root gene (BvSTI) that codes for a serine proteinase inhibitor. BvSTI is a root gene cloned from the F1016 breeding line that has moderate levels of resistance to the sugar beet root maggot ...

Sugar beet proteinase inhibitor (BvSTI) gene promoter is regulated by insects and wounding in transgenic Nicotiana benthamiana

USDA-ARS?s Scientific Manuscript database

A regulatory sequence from a serine proteinase inhibitor gene (BvSTIpro) shown to be up-regulated in resistant interactions with a root pest of sugar beet, the sugar beet root maggot, was fused to the ß-glucuronidase (GUS) reporter gene to characterize its expression patterns in transgenic Nicotiana...

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

PubMed

Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

2009-04-29

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Characterization and serology of the matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni before and after degradation by an endogenous proteinase.

PubMed

Eppstein, D A; Thoma, J A

1977-11-01

The intact matrix protein from a nuclear-polyhedrosis virus of the cabbage looper (Trichoplusia ni), isolated after inhibition of an endogenous serine-type proteinase, was further purified by molecular-sieve chromatography. The matrix protein was associated with carbohydrate moieties, and the carbohydrate content was determined for the two major peptides isolated after proteolysis by the endogenous proteinase. The association-dissociation interactions of the intact and proteinase-hydrolysed monomer units were characterized at high and low pH. At pH1.9, proteinase-degraded matrix protein dissociated into two different peptide fractions, FI and FII. Fraction FII, a single peptide of 9400 daltons, comprised one-third of the monomer unit of 28 000 daltons. At pH9.5, the degraded peptides were tightly associated in units equivalent to the intact monomer. These monomer equivalents associated to form a series of interconverting aggregates. The predominant aggregate sedimented at 11S and had a mol.wt greater than or equal to 200 000. Two non-cross-reacting antigens were present in the aggregate mixture. The presence of these two antigens does not reflect the presence of two different matrix proteins; rather, the expression of the antigens correlates with the degree of aggregation of the matrix protein.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

PubMed

He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

2008-10-01

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

Trichinella spiralis newborn larvae: characterization of a stage specific serine proteinase expression, NBL1, using monoclonal antibodies.

PubMed

Yang, Yong; Lacour, Sandrine A; Lainé-Prade, Véronique; Versillé, Nicolas; Grasset-Chevillot, Aurélie; Feng, Shuang; Liu, Ming Yuan; Boireau, Pascal; Vallée, Isabelle

2015-05-01

Trichinella spiralis is an intracellular parasitic nematode of mammalian skeletal muscle, causing a serious zoonotic disease in humans and showing a high economic impact mainly in pig breeding. Serine proteinases of T. spiralis play important roles in the host-parasite interactions mediating host invasion. In this study, we have focused on newborn larvae (NBL-1), the first identified serine proteinase from the NBL stage of T. spiralis. Five monoclonal antibodies (mAbs) directed against the C-terminal part of NBL1, were produced. These mAbs were IgG1κ isotype and specifically recognized as a common motif of 10 amino acids (PSSGSRPTYP). Selected mAbs were further characterized using antigens from various developmental stages of T. spiralis. Western blot revealed that selected mAbs reacted with the native NBL1 at Mr 50 kDa in the adult and NBL mixed antigens and NBL stage alone. Indirect immunofluorescence analysis revealed that selected mAbs intensely stained only the embryos within the gravid females and the NBL. Thus, the produced mAbs are useful tools for the characterization of NBL1 as a major antigen of Trichinella involved in the invasion of the host but also for the development of new serological tests with an early detection of T. spiralis infection.

Characterising the enzymatic profile of crude tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Cnidaria: Hydrozoa).

PubMed

Knittel, Paloma S; Long, Paul F; Brammall, Lucas; Marques, Antonio C; Almeida, Michelle T; Padilla, Gabriel; Moura-da-Silva, Ana M

2016-09-01

Jellyfish venoms are of medical and biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant in Olindias spp. High throughput mass spectrometry profiling of cnidarian venoms has become increasingly popular since the first description of the proteomic profile of putative toxins isolated from nematocysts of the hydrozoan jellyfish Olindias sambaquiensis describing the presence of orthologous enzymes as presented in venoms of advanced species as snakes. Rigorous bioinformatics analyses can aid functional annotation, but biochemical assays are prerequisite to unambiguously assign toxic function to a peptide or protein. Here we present results that experimentally confirm previously predicted proteomic analysis that crude venom extracts from tentacles of O. sambaquiensis are composed of polypeptides with metalloproteinase, serine proteinase and phospholipases A2 activities. Surprisingly, levels of serine proteinase and phospholipase A2 activities were comparable to those observed in venoms of Bothrops snakes which were used as positive controls in this study. Hence, these data offer new opportunities to explore serine proteinase and phospholipase A2 activities in the clinical sequelae following O. sambaquiensis envenomation, with future possible biopharmaceutical applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

PubMed Central

Pastar, Irena; Tonic, Ivana; Golic, Natasa; Kojic, Milan; van Kranenburg, Richard; Kleerebezem, Michiel; Topisirovic, Ljubisa; Jovanovic, Goran

2003-01-01

A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its αS1- and β-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca2+-free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase. PMID:14532028

Serine proteases in rodent hippocampus.

PubMed

Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

1998-09-04

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

AβPP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

PubMed Central

Xu, Feng; Previti, Mary Lou; Nieman, Marvin T.; Davis, Judianne; Schmaier, Alvin H.; Van Nostrand, William E.

2009-01-01

The amyloid β-protein precursor (AβPP) is best recognized as the precursor to the Aβ peptide that accumulates in the brains of patients with Alzheimer’s disease, but less is known about its physiological functions. Isoforms of AβPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AβPP regulates cerebral thrombosis in vivo (Xu et al., 2005 Proc. Natl. Acad. Sci. USA 102:18135–18140; Xu et al. 2007 Stroke 38:2598–2601). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AβPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AβPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model both AβPP−/− and APLP2−/− mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a pro-thrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage both AβPP−/− and APLP2−/− mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AβPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury. PMID:19403832

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

PubMed

Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

2001-01-01

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features.

PubMed

ten Have, Arjen; Dekkers, Ester; Kay, John; Phylip, Lowri H; van Kan, Jan A L

2004-07-01

Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1-5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.

Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Pan, Luqing; Hu, Fawen; Zheng, Debin

2011-09-01

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase inhibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured ( P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups, the hemocyte count reached the minimum in 3 h; granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macroglobulin-like activity reached the maximum in 3 h, and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

Impact of proteolytic enzymes in colorectal cancer development and progression.

PubMed

Herszényi, László; Barabás, Loránd; Hritz, István; István, Gábor; Tulassay, Zsolt

2014-10-07

Tumor invasion and metastasis is a highly complicated, multi-step phenomenon. In the complex event of tumor progression, tumor cells interact with basement membrane and extracellular matrix components. Proteolytic enzymes (proteinases) are involved in the degradation of extracellular matrix, but also in cancer invasion and metastasis. The four categories of proteinases (cysteine-, serine-, aspartic-, and metalloproteinases) are named and classified according to the essential catalytic component in their active site. We and others have shown that proteolytic enzymes play a major role not only in colorectal cancer (CRC) invasion and metastasis, but also in malignant transformation of precancerous lesions into cancer. Tissue and serum-plasma antigen concentrations of proteinases might be of great value in identifying patients with poor prognosis in CRC. Our results, in concordance with others indicate the potential tumor marker impact of proteinases for the early diagnosis of CRC. In addition, proteinases may also serve as potential target molecules for therapeutic agents.

Antibacterial activity of antileukoprotease.

PubMed Central

Hiemstra, P S; Maassen, R J; Stolk, J; Heinzel-Wieland, R; Steffens, G J; Dijkman, J H

1996-01-01

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection. PMID:8890201

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus.

PubMed

Macedo, Maria Lígia Rodrigues; Freire, Maria das Graças Machado; Franco, Octávio Luiz; Migliolo, Ludovico; de Oliveira, Caio Fernando Ramalho

2011-02-01

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests. Copyright © 2010 Elsevier Inc. All rights reserved.

Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis

PubMed Central

Wang, Guohao; Liu, Xudong; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2016-01-01

The Huntington’s disease (HD) protein, huntingtin (HTT), is essential for early development. Because suppressing the expression of mutant HTT is an important approach to treat the disease, we must first understand the normal function of Htt in adults versus younger animals. Using inducible Htt knockout mice, we found that Htt depletion does not lead to adult neurodegeneration or animal death at >4 mo of age, which was also verified by selectively depleting Htt in neurons. On the other hand, young Htt KO mice die at 2 mo of age of acute pancreatitis due to the degeneration of pancreatic acinar cells. Importantly, Htt interacts with the trypsin inhibitor, serine protease inhibitor Kazal-type 3 (Spink3), to inhibit activation of digestive enzymes in acinar cells in young mice, and transgenic HTT can rescue the early death of Htt KO mice. These findings point out age- and cell type-dependent vital functions of Htt and the safety of knocking down neuronal Htt expression in adult brains as a treatment. PMID:26951659

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

PubMed Central

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain. PMID:23724001

Identification of a serine proteinase homolog (Sp-SPH) involved in immune defense in the mud crab Scylla paramamosain.

PubMed

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05), and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05). Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

PubMed

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

Metastatic pancreatic adenocarcinoma associated with chronic calcific pancreatitis and a heterozygous SPINK1 N34S mutation.

PubMed

Moran, Robert A; Klapheke, Robert; Jalaly, Niloofar Y; Makary, Martin A; Hirose, Kenzo; Goggins, Michael; Wood, Laura; Laheru, Daniel A; Lennon, Anne Marie; Khashab, Mouen A; Singh, Vikesh K

2016-01-01

Contrary to patients with a cationic trypsinogen gene (PRSS1) mutations, Serine protease inhibitor Kazal-type 1 (SPINK1) heterozygote gene mutation carriers have a very low penetrance for acute, acute recurrent and/or chronic pancreatitis. Despite this, heterozygote SPINK 1 gene mutation patients have a similar age of onset of pancreatitis as PRSS 1 gene mutation patients. While the substantially elevated risk of pancreatic cancer in patients with PRSS1 gene mutations with chronic pancreatitis has been well established, little is known about the risk of pancreatic cancer in SPINK 1 gene mutation carriers with pancreatitis. We describe a case of malignant pancreatic cancer diagnosed in a young patient with chronic pancreatitis who is a SPINK 1 heterozygote gene mutation carrier. The risk of pancreatic cancer in gene mutation patients with chronic pancreatitis, in addition to screening options and management options for these patients is discussed. Copyright © 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

PubMed

Macedo-Ribeiro, Sandra; Almeida, Carla; Calisto, Bárbara M; Friedrich, Thomas; Mentele, Reinhard; Stürzebecher, Jörg; Fuentes-Prior, Pablo; Pereira, Pedro José Barbosa

2008-02-20

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Freshwater catfish jundiá (Rhamdia quelen) larvae are prepared to digest inert feed at the exogenous feeding onset: physiological and histological assessments.

PubMed

Silveira, Jenniffer; Silva, Carlos Peres; Cargnin-Ferreira, Eduardo; Alexandre, Daniel; Elias, Mariele Abádia; Fracalossi, Débora Machado

2013-12-01

This study assessed the morphological development of jundiá larvae's digestive system and digestive proteolytic activity. Specific serine proteinases activities varied over time, with the highest peak at 12 h after hatching (AH), which corresponded to 296.38 ± 84.20 mU mg⁻¹ for trypsin and 315.45 ± 42.16 mU mg⁻¹ for chymotrypsin. Specific aspartic proteinases activities increased up to the start of weaning, oscillated during that phase, but showed a consistent increase after that, resulting in the highest specific activity at 252 h AH (7.88 ± 0.68 mU mg⁻¹). Gel assays showed different molecular forms, especially of serine proteinases. Histology showed the gastrointestinal tract development onset at 0 h AH and open mouth at 4 h AH. At 16 h AH, the following differentiation of the digestive tract was evident: oropharyngeal cavity, esophagus, liver, pancreas, stomach, and intestine. At 40 h AH, zymogen granules in the pancreas were observed, and at 48 h AH, mucus in the digestive tract and gastric glands in the stomach. Findings indicate that jundiá has a functional stomach before the end of vitelline reserves. Therefore, jundiá larvae are probably capable to digest inert feed at the exogenous feeding onset.

Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.

PubMed

Wang, Zhen-Yu; Ding, Ling-Wen; Ge, Zhi-Juan; Wang, Zhaoyu; Wang, Fanghai; Li, Ning; Xu, Zeng-Fu

2007-01-01

SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses.

PubMed

Azouz, Nurit P; Ynga-Durand, Mario A; Caldwell, Julie M; Jain, Ayushi; Rochman, Mark; Fischesser, Demetria M; Ray, Leanne M; Bedard, Mary C; Mingler, Melissa K; Forney, Carmy; Eilerman, Matthew; Kuhl, Jonathan T; He, Hua; Biagini Myers, Jocelyn M; Mukkada, Vincent A; Putnam, Philip E; Khurana Hershey, Gurjit K; Kottyan, Leah C; Wen, Ting; Martin, Lisa J; Rothenberg, Marc E

2018-06-06

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5 ). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor-dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

Cassava Brown Streak Virus (Potyviridae) Encodes a Putative Maf/HAM1 Pyrophosphatase Implicated in Reduction of Mutations and a P1 Proteinase That Suppresses RNA Silencing but Contains No HC-Pro ▿

PubMed Central

Mbanzibwa, Deusdedith R.; Tian, Yanping; Mukasa, Settumba B.; Valkonen, Jari P. T.

2009-01-01

The complete positive-sense single-stranded RNA genome of Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae) was found to consist of 9,069 nucleotides and predicted to produce a polyprotein of 2,902 amino acids. It was lacking helper-component proteinase but contained a single P1 serine proteinase that strongly suppressed RNA silencing. Besides the exceptional structure of the 5′-proximal part of the genome, CBSV also contained a Maf/HAM1-like sequence (678 nucleotides, 226 amino acids) recombined between the replicase and coat protein domains in the 3′-proximal part of the genome, which is highly conserved in Potyviridae. HAM1 was flanked by consensus proteolytic cleavage sites for ipomovirus NIaPro cysteine proteinase. Homology of CBSV HAM1 with cellular Maf/HAM1 pyrophosphatases suggests that it may intercept noncanonical nucleoside triphosphates to reduce mutagenesis of viral RNA. PMID:19386713

Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors.

PubMed

Pyati, Prashant; Bandani, Ali R; Fitches, Elaine; Gatehouse, John A

2011-07-01

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Precursor processing for plant peptide hormone maturation by subtilisin-like serine proteinases.

PubMed

Schardon, Katharina; Hohl, Mathias; Graff, Lucile; Pfannstiel, Jens; Schulze, Waltraud; Stintzi, Annick; Schaller, Andreas

2016-12-23

Peptide hormones that regulate plant growth and development are derived from larger precursor proteins by proteolytic processing. Our study addressed the role of subtilisin-like proteinases (SBTs) in this process. Using tissue-specific expression of proteinase inhibitors as a tool to overcome functional redundancy, we found that SBT activity was required for the maturation of IDA (INFLORESCENCE DEFICIENT IN ABSCISSION), a peptide signal for the abscission of floral organs in Arabidopsis We identified three SBTs that process the IDA precursor in vitro, and this processing was shown to be required for the formation of mIDA (the mature and bioactive form of IDA) as the endogenous signaling peptide in vivo. Hence, SBTs act as prohormone convertases in plants, and several functionally redundant SBTs contribute to signal biogenesis. Copyright © 2016, American Association for the Advancement of Science.

Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung.

PubMed Central

Lee, J D; Kolattukudy, P E

1995-01-01

Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum for the enzymatic activity was 5.0 with elastin-Congo red as the substrate, and the activity was not significantly inhibited by pepstatin A, diazoacetyl norleucine methylester, and 1,2-epoxy-3-(p-nitrophenoxy) propane. The cDNA and gene encoding this aspartic proteinase were cloned and sequenced. The open reading frame, interrupted by three introns, would encode a protein of 393 amino acids composed of a putative 21-amino-acid signal peptide and a 49-amino-acid propeptide preceding the 323-amino-acid mature protein. The amino acid sequence of A. fumigatus aspartic proteinase has 70, 66, and 67% homology to the sequences of those from Aspergillus oryzae, Aspergillus awamori, and Aspergillus saitoi, respectively. The active-site motif (DTG) and the catalytic aspartic residues characteristic of aspartic proteinases are found in the presently described enzyme, indicating that it belongs to a family of aspartic proteinases. Polyclonal antibodies were produced in rabbits against both the mature and precursor forms of the aspartic proteinase expressed in Escherichia coli. Immunoblotting with both antibodies detected a 39-kDa mature enzyme in the culture supernatant of A. fumigatus. The aspartic proteinase activity was inhibited by the antibodies, suggesting that the aspartic proteinase in the culture supernatant corresponds to the product of the cloned gene. Immunogold electron microscopy showed that the aspartic proteinase was secreted by A. fumigatus invading neutropenic mouse lung and its secretion was directed toward the germ tubes of penetrating hyphae. PMID:7558282

Multiple forms of endopeptidase activity from jojoba seeds.

PubMed

Wolf, M J; Storey, R D

1990-01-01

The cotyledons of 27 day post-germination jojoba seedlings (Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their Mr and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and Mr of 58,000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and Mr of 41,000, 47,000 and 35,000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and Mr of 33,000.

Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus.

PubMed

Wu, Chenglin; Söderhäll, Irene; Kim, Young-A; Liu, Haipeng; Söderhäll, Kenneth

2008-10-01

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Caesalpinia bonduc serine proteinase inhibitor CbTI-2: Exploring the conformational features and antimalarial activity.

PubMed

Bhattacharyya, Arindam; Babu, C R

2017-10-01

Seeds of tropical legumes posses a repertoire of proteinase inhibitors (PI) and the current study highlights some structural/functional features of a strong serine PI from the seeds of Caesalpinia bonduc (CbTI-2). Following purification, N-terminal sequence of CbTI-2 revealed over 40% similarity with a few serine PIs of Caesalpinioideae subfamily. Upon exposure to metal ions and ionic/non ionic surfactants, CbTI-2 showed immense variation in the levels of antitryptic activity. Exposure of CbTI-2 to 1,4-Dithiothreitol, Guanidinium HCl, H 2 O 2 and Dimethyl sulfoxide led to a steady loss of inhibitory activity. Chemical modification of amino acids suggested an arginine as the active site residue. Circular Dichroism spectrum of native CbTI-2 revealed an unordered state. Secondary structure composition of CbTI-2 following exposure to extreme conditions (heat, acidic/alkaline environment, Guanidine hydrochloride and DTT) showed considerable perturbations that caused severe loss of antiproteolytic activity. DLS studies yielded a hydrodynamic radius of ∼2.2nm for CbTI-2 and also reconfirmed 1:1 stoichiometry for the trypsin-CbTI-2 complex. Initial studies indicated CbTI-2 to be a potent antiplasmodial agent by being highly toxic towards growth, schizont rupture process and erythrocytic invasion of Plasmodium falciparum. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

PubMed

Bilgin, M; Burgazli, K M; Rafiq, A; Mericliler, M; Neuhof, C; Oliva, M L; Parahuleva, M; Soydan, N; Doerr, O; Abdallah, Y; Erdogan, A

2014-01-01

Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

PubMed

de P G Gomes, Angélica; Dias, Simoni C; Bloch, Carlos; Melo, Francislete R; Furtado, José R; Monnerat, Rose G; Grossi-de-Sá, Maria F; Franco, Octávio L

2005-02-01

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

Negative regulation of prophenoloxidase (proPO) activation by a clip-domain serine proteinase homolog (SPH) from endoparasitoid venom.

PubMed

Zhang, Guangmei; Lu, Zhi-Qiang; Jiang, Haobo; Asgari, Sassan

2004-05-01

Most parasitic wasps inject maternal factors into the host hemocoel to suppress the host immune system and ensure successful development of their progeny. Melanization is one of the insect defence mechanisms against intruding pathogens or parasites. We previously isolated from the venom of Cotesia rubecula a 50 kDa protein that blocked melanization in the hemolymph of its host, Pieris rapae [Insect Biochem. Mol. Biol. 33 (2003) 1017]. This protein, designated Vn50, is a serine proteinase homolog (SPH) containing an amino-terminal clip domain. In this work, we demonstrated that recombinant Vn50 bound P. rapae hemolymph components that were recognized by antisera to Tenebrio molitor prophenoloxidase (proPO) and Manduca sexta proPO-activating proteinase (PAP). Vn50 is stable in the host hemolymph-it remained intact for at least 72 h after parasitization. Using M. sexta as a model system, we found that Vn50 efficiently down-regulated proPO activation mediated by M. sexta PAP-1, SPH-1, and SPH-2. Vn50 did not inhibit active phenoloxidase (PO) or PAP-1, but it significantly reduced the proteolysis of proPO. If recombinant Vn50 binds P. rapae proPO and PAP (as suggested by the antibody reactions), it is likely that the molecular interactions among M. sexta proPO, PAP-1, and SPHs were impaired by this venom protein. A similar strategy might be employed by C. rubecula to negatively impact the proPO activation reaction in its natural host.

An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C.

PubMed Central

Cowan, D A; Smolenski, K A; Daniel, R M; Morgan, H W

1987-01-01

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5. Images Fig. 2. Fig. 5. PMID:3120701

Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta.

PubMed

Hamza, Rim; Pérez-Hedo, Meritxell; Urbaneja, Alberto; Rambla, José L; Granell, Antonio; Gaddour, Kamel; Beltrán, José P; Cañas, Luis A

2018-01-25

Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

PubMed Central

Pfundt, R; van Ruissen, F; van Vlijmen-Willems, I M; Alkemade, H A; Zeeuwen, P L; Jap, P H; Dijkman, H; Fransen, J; Croes, H; van Erp, P E; Schalkwijk, J

1996-01-01

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases. PMID:8823304

Characterization of a serine proteinase homologous (SPH) in Chinese mitten crab Eriocheir sinensis.

PubMed

Qin, Chuanjie; Chen, Liqiao; Qin, Jian G; Zhao, Daxian; Zhang, Hao; Wu, Ping; Li, Erchao

2010-01-01

The serine protease homologous (SPH) is an important cofactor of prophenoloxidase-activating enzyme (PPAE). The gene of SPH of Chinese mitten crab Eriocheir sinensis (EsSPH) in hemocytes was cloned and characterized using reverse transcript polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The SPH cDNA consisted of 1386 bp with an open reading frame (ORF) encoded a protein of 378 amino acids, 154 bp 5'-untranslated region, and 95 bp 3'-untranslated region. Sequence comparisons against the GenBank database showed that EsSPH deduced amino acids had an overall identity to the gene of serine protease family from 41% to 70% of 15 invertebrate species. The protein had the structural characteristics of SPH, including the conserved six cysteine residues in the N-terminal clip domain and the functional activity (His157, Asp209, Gly311) in the C-terminal serine proteinase-like domain. To analyze the role of EsSPH in an acute infection, the temporal expression of the EsSPH gene after the Aeromonas hydrophila challenge was measured by real-time RT-PCR. The EsSPH transcripts in hemocytes significantly increased at 6 h, 12 h and 48 h over time after the A. hydrophila injection. This expression pattern shows that EsSPH has the potential to defend against invading microorganisms. The mRNA transcripts of EsSPH were detected in all tissues with the highest in the hepatopancreas. Interestingly, the mRNA transcripts of EsSPH and proPO were found in ova and expressed in oosperms, suggesting that the maternal transfer of EsSPH and proPO may exit in crab, but this warrants confirmation in further research.

Beta vulgaris L. serine proteinase inhibitor gene expression correlates to insect pest resistance in sugar beet

USDA-ARS?s Scientific Manuscript database

Analyzing genes that can be used for improving sugar beet resistance to the sugar beet root maggot (SBRM, Tetanops myopaeformis Roder), one of the most destructive insect pests of sugar beet in North America, was a major goal in our investigation. We report on the expression patterns of a sugar beet...

Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.

PubMed

Sakaguchi, Masayoshi; Takezawa, Makoto; Nakazawa, Rie; Nozawa, Kazutaka; Kusakawa, Taro; Nagasawa, Takeshi; Sugahara, Yasusato; Kawakita, Masao

2008-05-01

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40 degrees C in terms of k(cat) value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Changes in the distribution of type II transmembrane serine protease, TMPRSS2 and in paracellular permeability in IPEC-J2 cells exposed to oxidative stress.

PubMed

Paszti-Gere, Erzsebet; Barna, Reka Fanni; Kovago, Csaba; Szauder, Ipoly; Ujhelyi, Gabriella; Jakab, Csaba; Meggyesházi, Nóra; Szekacs, Andras

2015-04-01

The effect of oxidative stress on barrier integrity and localization of transmembrane serine proteinase 2 (TMPRSS2) were studied using porcine epithelial IPEC-J2 cells on membrane inserts. Increased paracellular permeability of FITC-dextran 4 kDa (fluorescence intensity 43,508 ± 2,391 versus 3,550 ± 759) and that of gentamicin (3.41 ± 0.06 % increase to controls) were measured parallel with the reduced transepithelial electrical resistance (23.3 ± 4.06 % decrease) of cell layers 6 h after 1 h 1 mM H2O2 treatment. The immunohistochemical localization of adherens junctional β-catenin was not affected by reactive oxygen species (ROS) up to 4 mM H2O2. Peroxide-triggered enhanced paracellular permeability of IPEC-J2 cell layer was accompanied by predominantly cytoplasmic occurrence of TMPRSS2 embedded in cell membrane under physiological conditions. These results support that ROS can influence paracellular gate opening via multifaceted mode of action without involvement of β-catenin redistribution in adherens junction. Altered distribution pattern of TMPRSS2 and relocalized transmembrane serine protease activity may contribute to weakening of epithelial barrier integrity under acute oxidative stress.

Independent Subtilases Expansions in Fungi Associated with Animals

PubMed Central

Muszewska, Anna; Taylor, John W.; Szczesny, Pawel; Grynberg, Marcin

2011-01-01

Many socially important fungi encode an elevated number of subtilisin-like serine proteases, which have been shown to be involved in fungal mutualisms with grasses and in parasitism of insects, nematodes, plants, other fungi, and mammalian skin. These proteins have endopeptidase activities and constitute a significant part of fungal secretomes. Here, we use comparative genomics to investigate the relationship between the quality and quantity of serine proteases and the ability of fungi to cause disease in invertebrate and vertebrate animals. Our screen of previously unexamined fungi allowed us to annotate and identify nearly 1000 subtilisin-containing proteins and to describe six new categories of serine proteases. Architectures of predicted proteases reveal novel combinations of subtilisin domains with other, co-occurring domains. Phylogenetic analysis of the most common clade of fungal proteases, proteinase K, showed that gene family size changed independently in fungi, pathogenic to invertebrates (Hypocreales) and vertebrates (Onygenales). Interestingly, simultaneous expansions in the S8 and S53 families of subtilases in a single fungal species are rare. Our analysis finds that closely related systemic human pathogens may not show the same gene family expansions, and that related pathogens and nonpathogens may show the same type of gene family expansion. Therefore, the number of proteases does not appear to relate to pathogenicity. Instead, we hypothesize that the number of fungal serine proteases in a species is related to the use of the animal as a food source, whether it is dead or alive. PMID:21727238

Polymorphisms of clip domain serine proteinase and serine proteinase homolog in the swimming crab Portunus trituberculatus and their association with Vibrio alginolyticus

NASA Astrophysics Data System (ADS)

Liu, Meng; Liu, Yuan; Hui, Min; Song, Chengwen; Cui, Zhaoxia

2017-03-01

Clip domain serine proteases (cSPs) and their homologs (SPHs) play an important role in various biological processes that are essential components of extracellular signaling cascades, especially in the innate immune responses of invertebrates. Here, polymorphisms of PtcSP and PtSPH from the swimming crab Portunus trituberculatus were investigated to explore their association with resistance/susceptibility to Vibrio alginolyticus. Polymorphic loci were identified using Clustal X, and characterized with SPSS 16.0 software, and then the significance of genotype and allele frequencies between resistant and susceptible stocks was determined by a χ 2 test. A total of 109 and 77 single nucleotide polymorphisms (SNPs) were identified in the genomic fragments of PtcSP and PtSPH, respectively. Notably, nearly half of PtSPH polymorphisms were found in the non-coding exon 1. Fourteen SNPs investigated were significantly associated with susceptibility/resistance to V. alginolyticus ( P <0.05). Among them, eight SNPs were observed in introns, and one synonymous, four non-synonymous SNPs and one ins-del were found in coding exons. In addition, five simple sequence repeats (SSRs) were detected in intron 3 of PtcSP. Although there was no statistically significant difference of allele frequencies, the SSRs showed different polymorphic alleles on the basis of the repeat number between resistant and susceptible stocks. After further validation, polymorphisms investigated here might be applied to select potential molecular markers of P. trituberculatus with resistance to V. alginolyticus.

Prophenoloxidase system and its role in shrimp immune responses against major pathogens.

PubMed

Amparyup, Piti; Charoensapsri, Walaiporn; Tassanakajon, Anchalee

2013-04-01

The global shrimp industry still faces various serious disease-related problems that are mainly caused by pathogenic bacteria and viruses. Understanding the host defense mechanisms is likely to be beneficial in designing and implementing effective strategies to solve the current and future pathogen-related problems. Melanization, which is performed by phenoloxidase (PO) and controlled by the prophenoloxidase (proPO) activation cascade, plays an important role in the invertebrate immune system in allowing a rapid response to pathogen infection. The activation of the proPO system, by the specific recognition of microorganisms by pattern-recognition proteins (PRPs), triggers a serine proteinase cascade, eventually leading to the cleavage of the inactive proPO to the active PO that functions to produce the melanin and toxic reactive intermediates against invading pathogens. This review highlights the recent discoveries of the critical roles of the proPO system in the shrimp immune responses against major pathogens, and emphasizes the functional characterizations of four major groups of genes and proteins in the proPO cascade in penaeid shrimp, that is the PRPs, serine proteinases, proPO and inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

PubMed

Gao, Gang; Xu, Xiao-Xia; Yu, Jing; Li, Lin-Miao; Ju, Wen-Yan; Jin, Feng-Liang; Freed, Shoaib

2016-09-01

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella. © 2016 Wiley Periodicals, Inc.

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization.

PubMed

Ferrer, Marvin; Rodriguez, Hilma; Zara, Lindsay; Yu, Yang; Xu, Wei; Oko, Richard

2012-09-01

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.

Matriptase Induction of Metalloproteinase‐Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms

PubMed Central

Wilkinson, David J.; Habgood, Angela; Lamb, Heather K.; Thompson, Paul; Hawkins, Alastair R.; Désilets, Antoine; Leduc, Richard; Steinmetzer, Torsten; Hammami, Maya; Lee, Melody S.; Craik, Charles S.; Watson, Sharon; Lin, Hua; Milner, Jennifer M.

2017-01-01

Objective To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. Methods Aggrecan release from human OA cartilage explants and human stem cell–derived cartilage discs was evaluated, and cartilage‐conditioned media were used for Western blotting. Gene expression was analyzed by real‐time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. Results The addition of soluble recombinant matriptase promoted a time‐dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease‐activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase– and aggrecanase‐mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low‐density lipoprotein receptor–related protein 1 (LRP‐1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase‐mediated aggrecan cleavage. Conclusion Matriptase potently induces the release of metalloproteinase‐generated aggrecan fragments as well as soluble LRP‐1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease‐modifying therapy in OA. PMID:28464560

The role of proteolytic enzymes in degradation of plant tissues: Summary report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewosz, J.; Kelman, A.; Sequeira, L.

1989-01-01

The proteolytic enzymes produced by Erwinia carotovora subsp. carotovora (Ecc-strain SR 394) grown on various media were examined by isoelectrofocusing in polyacrylamide gels over a pH range of 3-10. In addition to the main protease present in culture filtrates, low concentrations of several other proteases were present in extracts from potato tubers infected by Ecc. Proteases from all these sources were similar and had the following properties: pH optimum near 8.0, calcium dependent, insensitive to serine proteinase and SH-proteinase inhibitors, inhibited by EDTA, and highly thermostable. These enzymes degraded gelatin, soluble collagen and Hide Powder Azure, and showed weak activitymore » on casein, but did not degrade insoluble collagen or elastin.« less

Intracellular Localization and Trafficking of Serine Proteinase AhSub and Cysteine Proteinase AhCP of Acanthamoeba healyi

PubMed Central

Moon, E.-K.; Lee, S.-T.; Chung, D.-I.; Kong, H.-H.

2006-01-01

Proteinases have been proposed to play important roles in pathogenesis and various biologic actions in Acanthamoeba. Although genetic characteristics of several proteases of Acanthamoeba have been reported, the intracellular localization and trafficking of these enzymes has yet to be studied. In the present study, we analyzed the intracellular localization and trafficking of two proteinases, AhSub and AhCP, of Acanthamoeba healyi by transient transfection. Full-length AhSub-enhanced green fluorescent protein (EGFP) fusion protein was found in intracellular vesicle-like structures of transfected amoebae. Time-lapse photographs confirmed the secretion of the fluorescent material of the vesicle toward the extracellular space. The mutated AhSub, of which the pre or prepro region was deleted, was found to localize diffusely throughout the cytoplasm of the amoeba rather than concentrated in the secretory vesicle. Transfection of the construct containing the pre region only showed the same localization and trafficking of the full-length AhSub. A cysteine proteinase AhCP-EGFP fusion protein showed similar localization in the vesicle-like structure in the amoeba. However, using Lyso Tracker analysis, these vesicular structures of AhCP were confirmed to be lysosomes rather than secretory vesicles. The AhCP construct with a deletion of the prepro region showed a dispersed distribution of fluorescence in the cytoplasm of the cells. These results indicated that AhSub and AhCP would play different roles in Acanthameoba biology and that the pre region of AhSub and pro region of AhCP are important for proper intracellular localization and trafficking of each proteinase. PMID:16400174

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

PubMed Central

Headey, Stephen J.; MacAskill, Ursula K.; Wright, Michele A.; Claridge, Jolyon K.; Edwards, Patrick J. B.; Farley, Peter C.; Christeller, John T.; Laing, William A.; Pascal, Steven M.

2010-01-01

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp32–Asp215 diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin. PMID:20538608

An alternative easy method for antibody purification and analysis of protein-protein interaction using GST fusion proteins immobilized onto glutathione-agarose.

PubMed

Zalazar, L; Alonso, C A I; De Castro, R E; Cesari, A

2014-01-01

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.

Genetic and phenotypic heterogeneity in tropical calcific pancreatitis.

PubMed

Paliwal, Sumit; Bhaskar, Seema; Chandak, Giriraj R

2014-12-14

Tropical calcific pancreatitis (TCP) is a form of chronic non-alcoholic pancreatitis initially reported in the developing parts of the tropical world. The clinical phenotype of TCP has undergone marked changes since its first description in 1968. The disease is now seen in relatively older people with less severe symptoms. In addition, there are varying reports on the proportion of cases presenting with imaging abnormalities like calcification, ductal dilation, and glandular atrophy. Significant progress has also been made in understanding the etiopathology of TCP. The role of malnutrition and cassava toxicity in its pathogenesis is disproven and few studies have focused on the role of micronutrient deficiency and oxidative stress in the etiopathogenesis of TCP. Emerging evidence support an important role for genetic risk factors in TCP. Several studies have shown that, rather than mutations in trypsinogens, variants in serine protease inhibitor kazal type 1, cathepsin B, chymotrypsin C, cystic fibrosis transmembrane regulator, and carboxypeptidase A1, predict risk of TCP. These studies also provided evidence of mutational heterogeneity between TCP and chronic pancreatitis in Western populations. The current review summarizes recent advances that have implications in the understanding of the pathophysiology and thus, heterogeneity in genotype-phenotype correlations in TCP.

The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics

PubMed Central

2013-01-01

Background Snake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites. Results To better understand the evolutionary changes that resulted in the potent venom of a population of C. horridus from northern Florida, we sequenced the venom-gland transcriptome of an animal from this population for comparison with the previously described transcriptome of the eastern diamondback rattlesnake (C. adamanteus), a congener with a more typical rattlesnake venom. Relative to the toxin transcription of C. adamanteus, which consisted primarily of snake-venom metalloproteinases, C-type lectins, snake-venom serine proteinases, and myotoxin-A, the toxin transcription of C. horridus was far simpler in composition and consisted almost entirely of snake-venom serine proteinases, phospholipases A2, and bradykinin-potentiating and C-type natriuretic peptides. Crotalus horridus lacked significant expression of the hemorrhagic snake-venom metalloproteinases and C-type lectins. Evolution of shared toxin families involved differential expansion and loss of toxin clades within each species and pronounced differences in the highly expressed toxin paralogs. Toxin genes showed significantly higher rates of nonsynonymous substitution than nontoxin genes. The expression patterns of nontoxin genes were conserved between species, despite the vast differences in toxin expression. Conclusions Our results represent the first complete, sequence-based comparison between the venoms of closely related snake species and reveal in unprecedented detail the rapid evolution of snake venoms. We found that the difference in venom properties resulted from major changes in expression levels of toxin gene families, differential gene-family expansion and loss, changes in which paralogs within gene families were expressed at high levels, and higher nonsynonymous substitution rates in the toxin genes relative to nontoxins. These massive alterations in the genetics of the venom phenotype emphasize the evolutionary lability and flexibility of this ecologically critical trait. PMID:23758969

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

PubMed

Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

2012-03-01

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

PubMed Central

Huntington, J. A.; Patston, P. A.; Gettins, P. G.

1995-01-01

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7613461

The Contribution of Caseins to the Amino Acid Supply for Lactococcus lactis Depends on the Type of Cell Envelope Proteinase

PubMed Central

Flambard, Benedicte; Helinck, Sandra; Richard, Jean; Juillard, Vincent

1998-01-01

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase. PMID:9603805

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development[C][W

PubMed Central

Hartl, Markus; Giri, Ashok P.; Kaur, Harleen; Baldwin, Ian T.

2010-01-01

Solanaceaeous taxa produce diverse peptide serine proteinase inhibitors (SPIs), known antidigestive defenses that might also control endogenous plant proteases. If and how a plant coordinates and combines its different SPIs for the defense against herbivores and if these SPIs simultaneously serve developmental functions is unknown. We examine Solanum nigrum’s SPI profile, comprising four different active inhibitors, of which the most abundant proved to be novel, to understand their functional specialization in an ecological context. Transcript and activity characterization revealed tissue-specific and insect-elicited accumulation patterns. Stable and transient gene silencing of all four SPIs revealed different specificities for target proteinases: the novel SPI2c displayed high specificity for trypsin and chymotrypsin, while two other SPI2 homologs were highly active against subtilisin. In field and lab experiments, we found all four SPIs to display herbivore- and gene-specific defensive properties, with dissimilar effects on closely related species. However, we did not observe any clear developmental phenotype in SPI-silenced plants, suggesting that SPIs do not play a major role in regulating endogenous proteases under the conditions studied. In summary, specific single SPIs or their combinations defend S. nigrum against generalist herbivores, while the defense against herbivores specialized on SPI-rich diets requires other unknown defense mechanisms. PMID:21177479

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

PubMed Central

Kessenbrock, Kai; Fröhlich, Leopold; Sixt, Michael; Lämmermann, Tim; Pfister, Heiko; Bateman, Andrew; Belaaouaj, Azzaq; Ring, Johannes; Ollert, Markus; Fässler, Reinhard; Jenne, Dieter E.

2008-01-01

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents. PMID:18568075

Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases.

PubMed

Brand, G D; Pires, D A T; Furtado, J R; Cooper, A; Freitas, S M; Bloch, C

2017-03-15

The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteinase-activated receptor 2 (PAR(2)) in cholangiocarcinoma (CCA) cells: effects on signaling and cellular level.

PubMed

Kaufmann, Roland; Hascher, Alexander; Mussbach, Franziska; Henklein, Petra; Katenkamp, Kathrin; Westermann, Martin; Settmacher, Utz

2012-12-01

In this study, we demonstrate functional expression of the proteinase-activated receptor 2 (PAR(2)), a member of a G-protein receptor subfamily in primary cholangiocarcinoma (PCCA) cell cultures. Treatment of PCCA cells with the serine proteinase trypsin and the PAR(2)-selective activating peptide, furoyl-LIGRLO-NH(2), increased migration across a collagen membrane barrier. This effect was inhibited by a PAR(2)-selective pepducin antagonist peptide (P2pal-18S) and it was also blocked with the Met receptor tyrosine kinase (Met) inhibitors SU 11274 and PHA 665752, the MAPKinase inhibitors PD 98059 and SL 327, and the Stat3 inhibitor Stattic. The involvement of Met, p42/p44 MAPKinases and Stat3 in PAR(2)-mediated PCCA cell signaling was further supported by the findings that trypsin and the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated activating phosphorylation of these signaling molecules in cholangiocarcinoma cells. With our results, we provide a novel signal transduction module in cholangiocarcinoma cell migration involving PAR(2)-driven activation of Met, p42/p44 MAPKinases and Stat3.

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

PubMed

Schmidt, Karin; Gardill, Bernd R; Kern, Alina; Kirchweger, Peter; Börsch, Michael; Muller, Yves A

2018-05-14

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

Therapeutic targeting of SPINK1-positive prostate cancer.

PubMed

Ateeq, Bushra; Tomlins, Scott A; Laxman, Bharathi; Asangani, Irfan A; Cao, Qi; Cao, Xuhong; Li, Yong; Wang, Xiaoju; Feng, Felix Y; Pienta, Kenneth J; Varambally, Sooryanarayana; Chinnaiyan, Arul M

2011-03-02

Gene fusions involving ETS (erythroblastosis virus E26 transformation-specific) family transcription factors are found in ~50% of prostate cancers and as such can be used as a basis for the molecular subclassification of prostate cancer. Previously, we showed that marked overexpression of SPINK1 (serine peptidase inhibitor, Kazal type 1), which encodes a secreted serine protease inhibitor, defines an aggressive molecular subtype of ETS fusion-negative prostate cancers (SPINK1+/ETS⁻, ~10% of all prostate cancers). Here, we examined the potential of SPINK1 as an extracellular therapeutic target in prostate cancer. Recombinant SPINK1 protein (rSPINK1) stimulated cell proliferation in benign RWPE as well as cancerous prostate cells. Indeed, RWPE cells treated with either rSPINK1 or conditioned medium from 22RV1 prostate cancer cells (SPINK1+/ETS⁻) significantly increased cell invasion and intravasation when compared with untreated cells. In contrast, knockdown of SPINK1 in 22RV1 cells inhibited cell proliferation, cell invasion, and tumor growth in xenograft assays. 22RV1 cell proliferation, invasion, and intravasation were attenuated by a monoclonal antibody (mAb) to SPINK1 as well. We also demonstrated that SPINK1 partially mediated its neoplastic effects through interaction with the epidermal growth factor receptor (EGFR). Administration of antibodies to SPINK1 or EGFR (cetuximab) in mice bearing 22RV1 xenografts attenuated tumor growth by more than 60 and 40%, respectively, or ~75% when combined, without affecting PC3 xenograft (SPINK1⁻/ETS⁻) growth. Thus, this study suggests that SPINK1 may be a therapeutic target in a subset of patients with SPINK1+/ETS⁻ prostate cancer. Our results provide a rationale for both the development of humanized mAbs to SPINK1 and evaluation of EGFR inhibition in SPINK1+/ETS⁻ prostate cancers.

The Etiology and Clinical Course of Chronic Pancreatitis in Children With Early Onset of the Disease.

PubMed

Wejnarska, Karolina; Kolodziejczyk, Elwira; Wertheim-Tysarowska, Katarzyna; Dadalski, Maciej; Sobczynska-Tomaszewska, Agnieszka; Kierkus, Jarosław; Bal, Jerzy; Rygiel, Agnieszka Magdalena; Oracz, Grzegorz

2016-12-01

The etiological factors of chronic pancreatitis (CP) in children differ from those in adults. To date, no study has assessed the clinical course of CP in young children. The aim of our study was to evaluate the etiology and the clinical presentation of the disease in children with disease onset before 5 years of age in comparison to later-onset of CP. A total of 276 children with CP, hospitalized from 1988 to 2015, were enrolled in the study. Data on presentation, diagnostic findings, and treatment were reviewed. Two hundred sixty patients were screened for the most frequent mutations in major pancreatitis-associated genes, such as cationic trypsinogen/serine protease gene (PRSS1), serine protease inhibitor, Kazal type 1 gene (SPINK1), and cystic fibrosis transmembrane conductance regulator gene (CFTR). The disease onset before the age of 5 years occurred in 51 patients (group 1), the later onset in 225 patients (group 2). We found no significant discrepancies in distribution of the etiological factors between groups. The youngest patients (group 1) had more pancreatitis episodes (median 5.0 vs 3.00; P < 0.05) and underwent surgeries more frequently (25.5% vs 8.9%; P < 0.05). It could be associated with significantly longer follow-up in early onset group (median 6 vs 4 years; P < 0.05). There were no differences in nutritional status or exocrine and endocrine pancreatic function. Early- and later-onset pancreatitis have similar etiological factors with predominance of gene mutations. The most frequent mutation found was p.Asn34Ser (N34S) in SPINK1 gene. The clinical presentation differed in number of pancreatitis episodes and frequency of surgeries.

Targeted Gene Next-Generation Sequencing in Chinese Children with Chronic Pancreatitis and Acute Recurrent Pancreatitis.

PubMed

Xiao, Yuan; Yuan, Wentao; Yu, Bo; Guo, Yan; Xu, Xu; Wang, Xinqiong; Yu, Yi; Yu, Yi; Gong, Biao; Xu, Chundi

2017-12-01

To identify causal mutations in certain genes in children with acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP). After patients were enrolled (CP, 55; ARP, 14) and their clinical characteristics were investigated, we performed next-generation sequencing to detect nucleotide variations among the following 10 genes: cationic trypsinogen protease serine 1 (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator gene (CFTR), chymotrypsin C (CTRC), calcium-sensing receptor (CASR), cathepsin B (CTSB), keratin 8 (KRT8), CLAUDIN 2 (CLDN2), carboxypeptidase A1 (CPA1), and ATPase type 8B member 1 (ATP8B1). Mutations were searched against online databases to obtain information on the cause of the diseases. Certain novel mutations were analyzed using the SIFT2 and Polyphen-2 to predict the effect on protein function. There were 45 patients with CP and 10 patients with ARP who harbored 1 or more mutations in these genes; 45 patients had at least 1 mutation related to pancreatitis. Mutations were observed in the PRSS1, SPINK1, and CFTR genes in 17 patients, the CASR gene in 5 patients, and the CTSB, CTRC, and KRT8 genes in 1 patient. Mutations were not found in the CLDN, CPA1, or ATP8B1 genes. We found that mutations in SPINK1 may increase the risk of pancreatic duct stones (OR, 11.07; P = .003). The patients with CFTR mutations had a higher level of serum amylase (316.0 U/L vs 92.5 U/L; P = .026). Mutations, especially those in PRSS1, SPINK1, and CFTR, accounted for the major etiologies in Chinese children with CP or ARP. Children presenting mutations in the SPINK1 gene may have a higher risk of developing pancreatic duct stones. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization and expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

PubMed

Lin, Hailan; Lin, Xijian; Zhu, Jiwei; Yu, Xiao-Qiang; Xia, Xiaofeng; Yao, Fengluan; Yang, Guang; You, Minsheng

2017-02-14

Serine protease inhibitors (SPIs) have been found in all living organisms and play significant roles in digestion, development and innate immunity. In this study, we present a genome-wide identification and expression profiling of SPI genes in the diamondback moth, Plutella xylostella (L.), a major pest of cruciferous crops with global distribution and broad resistance to different types of insecticides. A total of 61 potential SPI genes were identified in the P. xylostella genome, and these SPIs were classified into serpins, canonical inhibitors, and alpha-2-macroglobulins based on their modes of action. Sequence alignments showed that amino acid residues in the hinge region of known inhibitory serpins from other insect species were conserved in most P. xylostella serpins, suggesting that these P. xylostella serpins may be functionally active. Phylogenetic analysis confirmed that P. xylostella inhibitory serpins were clustered with known inhibitory serpins from six other insect species. More interestingly, nine serpins were highly similar to the orthologues in Manduca sexta which have been demonstrated to participate in regulating the prophenoloxidase activation cascade, an important innate immune response in insects. Of the 61 P.xylostella SPI genes, 33 were canonical SPIs containing seven types of inhibitor domains, including Kunitz, Kazal, TIL, amfpi, Antistasin, WAP and Pacifastin. Moreover, some SPIs contained additional non-inhibitor domains, including spondin_N, reeler, and other modules, which may be involved in protein-protein interactions. Gene expression profiling showed gene-differential, stage- and sex-specific expression patterns of SPIs, suggesting that SPIs may be involved in multiple physiological processes in P. xylostella. This is the most comprehensive investigation so far on SPI genes in P. xylostella. The characterized features and expression patterns of P. xylostella SPIs indicate that the SPI family genes may be involved in innate immunity of this species. Our findings provide valuable information for uncovering further biological roles of SPI genes in P. xylostella.

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

PubMed Central

Silvestre, Frédéric; Linares-Casenave, Javier; Doroshov, Serge I.; Kültz, Dietmar

2012-01-01

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay / Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26°C and micro-injected with Seleno-L-Methionine to reach 8 μg g-1 selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P<0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline. PMID:20435339

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lund, Leif R; Romer, John; Thomasset, Nicole

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfatedmore » glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisonetreated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.« less

Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

PubMed Central

Wang, Yang; Lu, Zhiqiang; Jiang, Haobo

2014-01-01

Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta. PMID:24768974

Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

PubMed Central

Stapels, Daphne A. C.; Ramyar, Kasra X.; Bischoff, Markus; von Köckritz-Blickwede, Maren; Milder, Fin J.; Ruyken, Maartje; Eisenbeis, Janina; McWhorter, William J.; Herrmann, Mathias; van Kessel, Kok P. M.; Geisbrecht, Brian V.; Rooijakkers, Suzan H. M.

2014-01-01

Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity. PMID:25161283

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

PubMed

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes.

PubMed

Rokyta, Darin R; Wray, Kenneth P; Lemmon, Alan R; Lemmon, Emily Moriarty; Caudle, S Brian

2011-04-01

Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history. Copyright © 2011 Elsevier Ltd. All rights reserved.

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles.

PubMed

Janciauskiene, S

2001-03-26

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.

A clip-domain serine proteinase homolog (SPH) in oriental river prawn, Macrobrachium nipponense provides insights into its role in innate immune response.

PubMed

Ding, Zhili; Kong, Youqin; Chen, Liqiao; Qin, Jianguang; Sun, Shengming; Li, Ming; Du, Zhenyu; Ye, Jinyun

2014-08-01

In this study, a clip-domain serine proteinase homolog designated as MnSPH was cloned and characterized from a freshwater prawn Macrobrachium nipponense. The full-length cDNA of MnSPH was 1897 bp and contained a 1701 bp open reading frame (ORF) encoding a protein of 566 amino acids, a 103 bp 5'-untranslated region, and a 93 bp 3'-untranslated region. Sequence comparison showed that the deduced amino acids of MnSPH shared 30-59% identity with sequences reported in other animals. Tissue distribution analysis indicated that the MnSPH transcripts were present in all detected tissues with highest in the hepatopancreas and ovary. The MnSPH mRNA levels in the developing ovary were stable at the initial three developmental stages, then increased gradually from stage IV (later vitellogenesis), and reached a maximum at stage VI (paracmasis). Furthermore, the expression of MnSPH mRNA in hemocytes was significantly up-regulated at 1.5 h, 6 h, 12 h and 48 h post Aeromonas hydrophila injection. The increased phenoloxidase activity also demonstrated a clear time-dependent pattern after A. hydrophila challenge. These results suggest that MnSPH participates in resisting to pathogenic microorganisms and plays a pivotal role in host defense against microbe invasion in M. nipponense. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chemical Component and Proteomic Study of the Amphibalanus (= Balanus) amphitrite Shell

PubMed Central

Zhang, Gen; He, Li-sheng; Wong, Yue-Him; Xu, Ying; Zhang, Yu; Qian, Pei-yuan

2015-01-01

As typical biofoulers, barnacles possess hard shells and cause serious biofouling problems. In this study, we analyzed the protein component of the barnacle Amphibalanus (= Balanus) amphitrite shell using gel-based proteomics. The results revealed 52 proteins in the A. Amphitrite shell. Among them, 40 proteins were categorized into 11 functional groups based on KOG database, and the remaining 12 proteins were unknown. Besides the known proteins in barnacle shell (SIPC, carbonic anhydrase and acidic acid matrix protein), we also identified chorion peroxidase, C-type lectin-like domains, serine proteases and proteinase inhibitor proteins in the A. Amphitrite shell. The sequences of these proteins were characterized and their potential functions were discussed. Histology and DAPI staining revealed living cells in the shell, which might secrete the shell proteins identified in this study. PMID:26222041

Menstrual breakdown of human endometrium can be mimicked in vitro and is selectively and reversibly blocked by inhibitors of matrix metalloproteinases.

PubMed Central

Marbaix, E; Kokorine, I; Moulin, P; Donnez, J; Eeckhout, Y; Courtoy, P J

1996-01-01

The mechanisms underlying the menstrual lysis leading to shedding of the human endometrium and its accompanying bleeding are still largely unknown. In particular, whether breakdown of the endometrial fibrillar extra-cellular matrix that precedes bleeding depends on aspartic-, cysteine-, serine-, or metalloproteinases remains unclear. In the present study, menstrual regression of the human endometrium was mimicked in organ culture. Whereas sex steroids could preserve tissue integrity only in nonperimenstrual explants, matrix breakdown upon sex steroid deprivation was completely and reversibly inhibited at all stages of the menstrual cycle by specific inhibitors of matrix metalloproteinases, but not by inhibitors of the other classes of proteinases. Matrix metalloproteinases are thus identified as the key class of proteinases involved in the initiation of menstruation. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8799164

SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins.

PubMed

Pak, Stephen C; Kumar, Vasantha; Tsu, Christopher; Luke, Cliff J; Askew, Yuko S; Askew, David J; Mills, David R; Brömme, Dieter; Silverman, Gary A

2004-04-09

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as alpha(1)-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans.

Shrimp serine proteinase homologues PmMasSPH-1 and -2 play a role in the activation of the prophenoloxidase system.

PubMed

Jearaphunt, Miti; Amparyup, Piti; Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Senapin, Saengchan; Tassanakajon, Anchalee

2015-01-01

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system.

Virtual ligand screening of the National Cancer Institute (NCI) compound library leads to the allosteric inhibitory scaffolds of the West Nile Virus NS3 proteinase.

PubMed

Shiryaev, Sergey A; Cheltsov, Anton V; Gawlik, Katarzyna; Ratnikov, Boris I; Strongin, Alex Y

2011-02-01

Viruses of the genus Flavivirus are responsible for significant human disease and mortality. The N-terminal domain of the flaviviral nonstructural (NS)3 protein codes for the serine, chymotrypsin-fold proteinase (NS3pro). The presence of the nonstructural (NS)2B cofactor, which is encoded by the upstream gene in the flaviviral genome, is necessary for NS3pro to exhibit its proteolytic activity. The two-component NS2B-NS3pro functional activity is essential for the viral polyprotein processing and replication. Both the structure and the function of NS2B-NS3pro are conserved in the Flavivirus family. Because of its essential function in the posttranslational processing of the viral polyprotein precursor, NS2B-NS3pro is a promising target for anti-flavivirus drugs. To identify selective inhibitors with the reduced cross-reactivity and off-target effects, we focused our strategy on the allosteric inhibitors capable of targeting the NS2B-NS3pro interface rather than the NS3pro active site. Using virtual ligand screening of the diverse, ∼275,000-compound library and the catalytic domain of the two-component West Nile virus (WNV) NS2B-NS3pro as a receptor, we identified a limited subset of the novel inhibitory scaffolds. Several of the discovered compounds performed as allosteric inhibitors and exhibited a nanomolar range potency in the in vitro cleavage assays. The inhibitors were also potent in cell-based assays employing the sub-genomic, luciferase-tagged WNV and Dengue viral replicons. The selectivity of the inhibitors was confirmed using the in vitro cleavage assays with furin, a human serine proteinase, the substrate preferences of which are similar to those of WNV NS2B-NS3pro. Conceptually, the similar in silico drug discovery strategy may be readily employed for the identification of inhibitors of other flaviviruses.

Shrimp Serine Proteinase Homologues PmMasSPH-1 and -2 Play a Role in the Activation of the Prophenoloxidase System

PubMed Central

Jearaphunt, Miti; Amparyup, Piti; Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Senapin, Saengchan; Tassanakajon, Anchalee

2015-01-01

Melanization mediated by the prophenoloxidase (proPO) activating system is a rapid immune response used by invertebrates against intruding pathogens. Several masquerade-like and serine proteinase homologues (SPHs) have been demonstrated to play an essential role in proPO activation in insects and crustaceans. In a previous study, we characterized the masquerade-like SPH, PmMasSPH1, in the black tiger shrimp Penaeus monodon as a multifunctional immune protein based on its recognition and antimicrobial activity against the Gram-negative bacteria Vibrio harveyi. In the present study, we identify a novel SPH, known as PmMasSPH2, composed of an N-terminal clip domain and a C-terminal SP-like domain that share high similarity to those of other insect and crustacean SPHs. We demonstrate that gene silencing of PmMasSPH1 and PmMasSPH2 significantly reduces PO activity, resulting in a high number of V. harveyi in the hemolymph. Interestingly, knockdown of PmMasSPH1 suppressed not only its gene transcript but also other immune-related genes in the proPO system (e.g., PmPPAE2) and antimicrobial peptides (e.g., PenmonPEN3, PenmonPEN5, crustinPm1 and Crus-likePm). The PmMasSPH1 and PmMasSPH2 also show binding activity to peptidoglycan (PGN) of Gram-positive bacteria. Using a yeast two-hybrid analysis and co-immunoprecipitation, we demonstrate that PmMasSPH1 specifically interacted with the final proteinase of the proPO cascade, PmPPAE2. Furthermore, the presence of both PmMasSPH1 and PmPPAE2 enhances PGN-induced PO activity in vitro. Taken together, these results suggest the importance of PmMasSPHs in the activation of the shrimp proPO system. PMID:25803442

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

PubMed Central

Horwitz, Marshall S.; Jenne, Dieter E.; Gauthier, Francis

2010-01-01

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies. PMID:21079042

Proteolytic Activation Transforms Heparin Cofactor II into a Host Defense Molecule

PubMed Central

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M.; Malmsten, Martin; Mörgelin, Matthias

2013-01-01

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity. PMID:23656734

Proteolytic activation transforms heparin cofactor II into a host defense molecule.

PubMed

Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

2013-06-15

The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

PubMed Central

2013-01-01

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

DOE PAGES

Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; ...

2016-03-23

α 1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based onmore » biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography. PLOS« less

The role of fungal proteinases in pathophysiology of Stachybotrys chartarum.

PubMed

Yike, Iwona; Rand, Thomas; Dearborn, Dorr G

2007-10-01

The adverse health effects of Stachybotrys chartarum have often been linked to exposure to the trichothecene mycotoxins. Recent studies have shown that in addition to mycotoxins this fungus is capable of producing and secreting in vivo proteins such as hemolysins and proteinases. Spore extracts obtained from a high trichothecene producing isolate JS 58-17 exhibited a significantly lower proteolytic activity compared to the low trichothecene producer, JS 58-06. Growing isolates on rice or potato dextrose agar results in higher proteolytic activity of the spores compared to those grown on drywall. Proteinases in the spore extracts can hydrolyze gelatin and collagen I and IV. Analysis of zymograms shows the presence of several proteins with proteolytic activity in the spores of S. chartarum. Human tracheal epithelial cells exposed to spore extracts produced significantly higher levels of IL-6, IL-8, and TNF-alpha than control cells. This stimulation of cytokine production was completely abolished by Pefabloc, a serine protease inhibitor. Neutrophil numbers and proinflammatory cytokine (IL1-beta and TNF-alpha) concentrations were highly elevated in the lungs of 7 day old rat pups exposed intratracheally to 4 x 10(4) spores/gm body weight compared to control. No significant differences in those inflammatory indices in vivo were noted between the treatments with the high trichothecene producer, isolate JS 58-17 and JS 58-06, which does not produce macrocyclic trichothecenes. Immunohistochemistry revealed reduced collagen IV labeling in spore-induced lung granulomas in rat pups exposed to both isolates. These results suggest that proteinases from S. chartarum spores significantly contribute to lung inflammation and injury.

Potential of the Lectin/Inhibitor Isolated from Crataeva tapia Bark (CrataBL) for Controlling Callosobruchus maculatus Larva Development.

PubMed

Nunes, Natalia N S; Ferreira, Rodrigo S; Silva-Lucca, Rosemeire A; de Sá, Leonardo F R; de Oliveira, Antônia Elenir A; Correia, Maria Tereza dos S; Paiva, Patrícia Maria G; Wlodawer, Alexander; Oliva, Maria Luiza V

2015-12-09

Callosobruchus maculatus is an important predator of cowpeas. Due to infestation during storage, this insect affects the quality of seed and crop yield. This study aimed to investigate the effects of CrataBL, a multifunction protein isolated from Crataeva tapia bark, on C. maculatus larva development. The protein, which is stable even in extreme pH conditions, showed toxic activity, reducing the larval mass 45 and 70% at concentrations of 0.25 and 1.0% (w/w), respectively. Acting as an inhibitor, CrataBL decreased by 39% the activity of cysteine proteinases from larval gut. Conversely, the activity of serine proteinases was increased about 8-fold. The toxic properties of CrataBL may also be attributed to its capacity of binding to glycoproteins or glycosaminoglycans. Such binding interferes with larval metabolism, because CrataBL-FITC was found in the fat body, Malpighian tubules, and feces of larvae. These results demonstrate the potential of this protein for controlling larva development.

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

PubMed

Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M

2009-03-01

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T(m) approximately +1.5 degrees C) of the truncated enzyme (VPRDeltaC) as compared to the wild type VPR. Introduction of the N15D substitution into VPRDeltaC improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.

Phase diagram of crystallization of Aspergillus niger acid proteinase A, a non-pepsin-type acid proteinase

NASA Astrophysics Data System (ADS)

Kudo, Norio; Ataka, Mitsuo; Sasaki, Hiroshi; Muramatsu, Tomonari; Katsura, Tatsuo; Tanokura, Masaru

1996-10-01

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase with an extremely low isoelectric point (pI 3.3). The protein is crystallized from ammonium sulfate solutions of pH lower than 4. The crystallization is affected by the presence of dimethylsulfoxide (DMSO). We have studied the phase diagram of the crystallization of proteinase A in the absence and presence of DMSO, to clarify crystallization at such an extremely low pH and to study the effects of DMSO. The results indicate that the logarithm of protein solubility is a rectilinear function of ammonium sulfate concentration in both the absence and presence of DMSO. DMSO definitely lowers the solubility at relatively low concentrations of ammonium sulfate, but had little effect on protein solubility at higher concentrations of ammonium sulfate.

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

PubMed Central

Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

PubMed

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Polymorphism of alpha-1-antitrypsin in hematological malignancies

PubMed Central

2009-01-01

Alpha-1-antitrypsin (AAT) or serine protease inhibitor A1 (SERPINA1) is an important serine protease inhibitor in humans. The main physiological role of AAT is to inhibit neutrophil elastase (NE) released from triggered neutrophils, with an additional lesser role in the defense against damage inflicted by other serine proteases, such as cathepsin G and proteinase 3. Although there is a reported association between AAT polymorphism and different types of cancer, this association with hematological malignancies (HM) is, as yet, unknown. We identified AAT phenotypes by isoelectric focusing (in the pH 4.2-4.9 range) in 151 serum samples from patients with HM (Hodgkins lymphomas, non-Hodgkins lymphomas and malignant monoclonal gammopathies). Healthy blood-donors constituted the control group (n = 272). The evaluated population of patients as well as the control group, were at Hardy-Weinberg equilibrium for the AAT gene (χ2 = 4.42, d.f.11, p = 0.96 and χ2 = 4.71, d.f.11, p = 0.97, respectively). There was no difference in the frequency of deficient AAT alleles (Pi Z and Pi S) between patients and control. However, we found a significantly higher frequency of PiM1M1 homozygote and PiM1 allele in HM patients than in control (for phenotype: f = 0.5166 and 0.4118 respectively, p = 0.037; for allele: f = 0.7020 and 0.6360 respectively, p = 0.05). In addition, PiM homozygotes in HM-patients were more numerous than in controls (59% and 48%, respectively, p = 0.044). PiM1 alleles and PiM1 homozygotes are both associated with hematological malignancies, although this is considered a functionally normal AAT variant. PMID:21637443

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix.

PubMed

Appert-Collin, Aline; Bennasroune, Amar; Jeannesson, Pierre; Terryn, Christine; Fuhrmann, Guy; Morjani, Hamid; Dedieu, Stéphane

2017-07-04

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.

Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

PubMed

Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M

2003-06-01

The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection.

PubMed

Kaewkascholkul, Napol; Somboonviwat, Kulwadee; Asakawa, Shuichi; Hirono, Ikuo; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

2016-07-01

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response. Copyright © 2016. Published by Elsevier Ltd.

Porphyromonas gingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gliding motility

PubMed Central

Nakayama, K

2015-01-01

Porphyromonas gingivalis is a gram-negative, non-motile, anaerobic bacterium implicated as a major pathogen in periodontal disease. P. gingivalis grows as black-pigmented colonies on blood agar, and many bacteriologists have shown interest in this property. Studies of colonial pigmentation have revealed a number of important findings, including an association with the highly active extracellular and surface proteinases called gingipains that are found in P. gingivalis. The Por secretion system, a novel type IX secretion system (T9SS), has been implicated in gingipain secretion in studies using non-pigmented mutants. In addition, many potent virulence proteins, including the metallocarboxypeptidase CPG70, 35 kDa hemin-binding protein HBP35, peptidylarginine deiminase PAD and Lys-specific serine endopeptidase PepK, are secreted through the T9SS. These findings have not been limited to P. gingivalis but have been extended to other bacteria belonging to the phylum Bacteroidetes. Many Bacteroidetes species possess the T9SS, which is associated with gliding motility for some of these bacteria. PMID:25546073

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

PubMed Central

1986-01-01

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties. PMID:3537191

Template-dependent polypeptide synthesis in a factor- and energy-free translation system promoted by pyridine.

PubMed

Nitta, I; Ueda, T; Nojima, T; Watanabe, K

1995-10-01

We demonstrate here that a high concentration (40-70%) of pyridine, an aromatic tertiary amine catalyst, is able to promote translation on ribosomes without the presence of soluble protein factors or chemical energy sources. Compared with Monro's fragment reaction [Methods Enzymol. 20, 472-481 (1971)] which reflects only the peptidyltransferase step, this novel translation system can produce polypeptides with chain lengths of at least several tens of residues depending on the template RNA. In the presence of 60% pyridine, poly(U) and poly(UC) promoted incorporation of the respective amino acids, phenylalanine and serine-leucine, twofold, whereas poly(A) promoted the incorporation of lysine by only 25%. The degrees of polymerization of phenylalanine and lysine were up to the decamer and around 40mer, respectively. In poly(UC)-dependent oligo(serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products. This novel pyridine system evidently differs from the non-enzymatic translation system reported by Gavrilova and Spirin [FEBS Lett. 17, 324-326 (1971)]; the former system displays partial resistance toward deproteinization reagents such as SDS and proteinase K, whereas the latter system is completely sensitive.

Effect of sorghum type and malting on production of free amino nitrogen in conjunction with exogenous protease enzymes.

PubMed

Dlamini, Bhekisisa C; Buys, Elna M; Taylor, John R N

2015-01-01

Sorghum types suitable for brewing and bioethanol production are required. The effect of sorghum type (white non-tannin versus white type II tannin) on free amino nitrogen (FAN) production from sorghum grain and malt using exogenous protease enzymes was investigated over extended incubation at moderate temperature (45 °C). With grain in the absence of exogenous proteases, white non-tannin sorghum produced substantially higher levels of FAN than white type II tannin sorghum, due to the tannins in the latter. Incubating sorghum grain with neutral proteinase and amino-peptidase in combination improved FAN production. The two sorghum types produced similar FAN levels when malted and incubated in the absence of the exogenous proteases. When both sorghums were malted and incubated with neutral proteinase alone substantially more FAN yield (124-126 mg 100 g(-1)) occurred than with grains (61-84 mg 100 g(-1)). The combination of amino-peptidase and proteinase did not improve FAN further. Neither, did malting influence wort free amino acid profile. Group B amino acids constituted the highest percentage (42-47%). With grain, white non-tannin sorghum plus proteinase and amino-peptidase yields the highest FAN, with malt both white non-tannin and white type II tannin sorghums plus proteinase yield the highest FAN. © 2014 Society of Chemical Industry.

A Venom Serpin Splicing Isoform of the Endoparasitoid Wasp Pteromalus puparum Suppresses Host Prophenoloxidase Cascade by Forming Complexes with Host Hemolymph Proteinases*

PubMed Central

Yan, Zhichao; Fang, Qi; Liu, Yang; Xiao, Shan; Yang, Lei; Wang, Fei; An, Chunju; Werren, John H.; Ye, Gongyin

2017-01-01

To ensure successful parasitism, parasitoid wasps inject venom along with their eggs into their hosts. The venom serves to suppress host immune responses, including melanization. Venom from Pteromalus puparum, a pupal endoparasitoid, inhibits melanization of host hemolymph in vitro in a dose-dependent manner. Using assay-guided fractionation, a serpin splicing isoform with phenoloxidase inhibitory activity was identified as P. puparum serpin-1, venom isoform (PpS1V). This serpin gene has 16 predicted splicing isoforms that differ only in the C-terminal region. RT-PCR results show that the specific serpin isoform is differentially expressed in the venom gland. Recombinant PpS1V (rPpS1V) suppresses host prophenoloxidase (PPO) activation rather than inhibiting the phenoloxidase directly. Pulldown assays show that PpS1V forms complexes with two host hemolymph proteins, here named Pieris rapae hemolymph proteinase 8 (PrHP8) and P. rapae prophenoloxidase-activating proteinase 1 (PrPAP1), based on gene sequence blasting and phylogenetic analysis. The role of rPrPAP1 in the PPO activation cascade and its interaction with rPpS1V were confirmed. The stoichiometry of inhibition of PrPAP1 by PpS1V is 2.3. PpS1V also inhibits PPO activation in a non-natural host, Ostrinia furnacalis, through forming a complex with O. furnacalis serine protease 13 (OfSP13), an ortholog to PrPAP1. Our results identify a venom-enriched serpin isoform in P. puparum that inhibits host PPO activation, probably by forming a complex with host hemolymph proteinase PrPAP1. PMID:27913622

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

PubMed Central

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2013-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth. PMID:23527883

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

PubMed

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2014-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

A Trypanosoma cruzi-secreted 80 kDa proteinase with specificity for human collagen types I and IV.

PubMed Central

Santana, J M; Grellier, P; Schrével, J; Teixeira, A R

1997-01-01

Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy. PMID:9224638

Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

PubMed

Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

2015-10-01

Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients.

Enhancement of lymphocyte proliferation by mouse glandular kallikrein.

PubMed

Hu, Z Q; Murakami, K; Ikigai, H; Shimamura, T

1992-03-01

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans.

PubMed

Wang, Xuyong; Ribeiro, José M C; Broce, Alberto B; Wilkerson, Melinda J; Kanost, Michael R

2009-09-01

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen 5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental Tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.

An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans

PubMed Central

Wang, Xuyong; Ribeiro, José M. C.; Broce, Alberto B.; Wilkerson, Melinda J.; Kanost, Michael R.

2009-01-01

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen-5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls. PMID:19576987

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Chronic pancreatitis associated with the p.G208A variant of PRSS1 gene in a European patient.

PubMed

Hegyi, Eszter; Cierna, Iveta; Vavrova, Ludmila; Ilencikova, Denisa; Konecny, Michal; Kovacs, Laszlo

2014-01-10

The major etiologic factor of chronic pancreatitis in adults is excessive alcohol consumption, whereas among children structural anomalies, systemic and metabolic disorders, and genetic factors are prevalent. Mutations in the cationic trypsinogen gene (PRSS1) cause hereditary pancreatitis, while mutations in serine protease inhibitor Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR) and chymotrypsin C (CTRC) genes have been shown to associate with chronic pancreatitis as independent risk factors. We present a case of 13-year-old boy with idiopathic chronic pancreatitis. Given the unexplained attacks of pancreatitis since early childhood and despite the negative family history, molecular-genetic analysis of four pancreatitis susceptibility genes (PRSS1, SPINK1, CTRC and CFTR) was performed. The boy was found to carry the c.623G>C (p.G208A) mutation of the PRSS1 gene and the c.180C>T (p.G60G) mutation of the CTRC gene, both in heterozygous state. These mutations are considered as contributing risk factors for chronic pancreatitis. In children with idiopathic chronic pancreatitis genetic causes should be considered, even in absence of positive family history. To the best of our knowledge, this is the first description of a European patient with chronic pancreatitis associated with the p.G208A mutation of PRSS1 gene. This mutation was previously reported only in Asian subjects and is thought to be a unique genetic cause of pancreatitis in Asia.

The SPINK gene family and celiac disease susceptibility.

PubMed

Wapenaar, Martin C; Monsuur, Alienke J; Poell, Jos; van 't Slot, Ruben; Meijer, Jos W R; Meijer, Gerrit A; Mulder, Chris J; Mearin, Maria Luisa; Wijmenga, Cisca

2007-05-01

The gene family of serine protease inhibitors of the Kazal type (SPINK) are functional and positional candidate genes for celiac disease (CD). Our aim was to assess the gut mucosal gene expression and genetic association of SPINK1, -2, -4, and -5 in the Dutch CD population. Gene expression was determined for all four SPINK genes by quantitative reverse-transcription polymerase chain reaction in duodenal biopsy samples from untreated (n=15) and diet-treated patients (n=31) and controls (n=16). Genetic association of the four SPINK genes was tested within a total of 18 haplotype tagging SNPs, one coding SNP, 310 patients, and 180 controls. The SPINK4 study cohort was further expanded to include 479 CD cases and 540 controls. SPINK4 DNA sequence analysis was performed on six members of a multigeneration CD family to detect possible point mutations or deletions. SPINK4 showed differential gene expression, which was at its highest in untreated patients and dropped sharply upon commencement of a gluten-free diet. Genetic association tests for all four SPINK genes were negative, including SPINK4 in the extended case/control cohort. No SPINK4 mutations or deletions were observed in the multigeneration CD family with linkage to chromosome 9p21-13 nor was the coding SNP disease-specific. SPINK4 exhibits CD pathology-related differential gene expression, likely derived from altered goblet cell activity. All of the four SPINK genes tested do not contribute to the genetic risk for CD in the Dutch population.

Inhibitors of Eicosanoid Biosynthesis Influencing the Transcripts Level of sHSP21.4 Gene Induced by Pathogen Infections, in Antheraea pernyi

PubMed Central

Zhang, Congfen; Dai, Lishang; Wang, Lei; Qian, Cen; Wei, Guoqing; Li, Jun; Zhu, Baojian; Liu, Chaoliang

2015-01-01

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi response to microorganisms, a sHsp gene termed as Ap-sHSP21.4, was identified. This gene encoded a 21.4 kDa protein which shares the conserved structure of insect sHsps and belongs to sHSP21.4 family. Ap-sHSP21.4 was highly expressed in fat body and up-regulated in midgut and fat body of A. pernyi challenged with Escherichia coli, Beauveria bassiana and nuclear polyhedrosis virus (NPV), which was determined by quantitative real-time PCR. Meanwhile, knock down of Ap-sHSP21.4 with dsRNA result in the decrease at the expression levels of several immune response-related genes (defensin, Dopa decarboxylase, Toll1, lysozyme and Kazal-type serine protease inhibitor). Additionally, the impact of eicosanoid biosynthesis on the expression of Ap-sHSP21.4 response to NPV was determined using qPCR, inhibitors of eicosanoid biosynthesis significantly suppress Ap-HSP21.4 expression upon NPV challenge. All together, Ap-sHSP21.4 was involved in the immunity of A. pernyi against microorganism and possibly mediated by eicosanoids pathway. These results will shed light in the understanding of the pathogen-host interaction in A. pernyi. PMID:25844646

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.

PubMed

Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C

1997-02-01

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis.

PubMed

Macedo, Maria Lígia R; Freire, Maria das Graças M; Kubo, Carlos Eduardo G; Parra, José Roberto P

2011-01-01

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. Copyright © 2010 Elsevier Inc. All rights reserved.

Production and biochemical characterization of insecticidal enzymes from Aspergillus fumigatus toward Callosobruchus maculatus.

PubMed

Pereira, Jackeline L; Franco, Octávio L; Noronha, Eliane F

2006-06-01

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM: phenylmethylsulfonyl fluoride (100%) and 50 microM leupeptin (67.94%).

Proteolytic activities in yeast after UV irradiation. II. Variation in proteinase levels in mutants blocked in DNA-repair pathways.

PubMed

Schwencke, J; Moustacchi, E

1982-01-01

When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains. It is obvious that the induction of protease B activity following UV-treatment in Saccharomyces cannot be equated to the induction of the recA protein in Escherichia coli. However the correlation found between the block in mutagenic repair and the lack of UV-induction of protease B activity leads to questions on the possible role of certain protease activities in mutagenic repair in eucaryotic cells.

Deciphering the main venom components of the ectoparasitic ant-like bethylid wasp, Scleroderma guani.

PubMed

Zhu, Jia-Ying

2016-04-01

Similar to venom found in most venomous animals, parasitoid venoms contain a complex cocktail of proteins with potential agrichemical and pharmaceutical use. Even though parasitoids are one of the largest group of venomous animals, little is known about their venom composition. Recent few studies revealed high variated venom composition existing not only in different species but also between closely related strains, impling that increasing information on the venom proteins from more greater diversity of species of different taxa is key to comprehensively uncover the complete picture of parasitoid venom. Here, we explored the major protein components of the venom of ectoparasitic ant-like bethylid wasp, Scleroderma guani by an integrative transcriptomic-proteomic approach. Illumina deep sequencing of venom apparatus cDNA produced 49,873 transcripts. By mapping the peptide spectral data derived from venom reservoir against these transcripts, mass spectrometry analysis revealed ten main venom proteins, including serine proteinase, metalloprotease, dipeptidyl peptidase IV, esterase, antithrombin-III, acid phosphatase, neural/ectodermal development factor IMP-L2 like protein, venom allergen 3, and unknown protein. Interestingly, one serine proteinase was firstly identified with rarely high molecular weight about 200 kDa in parasitoid venom. The occurrence of abundant acid phosphatase, antithrombin-III and venom allergen 3 demonstrated that S. guani venom composition is similar to that of social wasp venoms. All identified venom genes showed abundantly biased expression in venom apparatus, indicating their virulent functions involved in parasitization. This study shed light on the more better understanding of parasitoid venom evolution across species and will facilitate the further elucidation of function and toxicity of these venom proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human eosinophils constitutively express a unique serine protease, PRSS33.

PubMed

Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

2017-07-01

Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Isolation, gene cloning and expression profile of a pathogen recognition protein: a serine proteinase homolog (Sp-SPH) involved in the antibacterial response in the crab Scylla paramamosain.

PubMed

Liu, Hai-peng; Chen, Rong-yuan; Zhang, Min; Wang, Ke-jian

2010-07-01

To identify the frontline defense molecules against microbial infection in the crab Scylla paramamosain, a live crab pathogenic microbe, Vibrio parahaemolyticus, was recruited as an affinity matrix to isolate innate immune factors from crab hemocytes lysate. Interestingly, a serine proteinase homolog (Sp-SPH) was obtained together with an antimicrobial peptide-antilipopolysaccharide factor (Sp-ALF). We then determined the full-length cDNA sequence of Sp-SPH, which contained 1298bp with an open reading frame of 1107bp encoding 369 amino acid residues. Multiple alignment analysis showed that the deduced amino acid sequences of Sp-SPH shared overall identity (83.8%) with those of SPH-containing proteins from other crab species. Tissue distribution analysis indicated that the Sp-SPH transcripts were present in various tissues including eye stalk, subcuticular epidermis, gill, hemocyte, stomach, thorax ganglion, brain and muscle of S. paramamosain. The Sp-SPH was highly expressed in selected different development stages including embryo (I, II, III and V), zoea (I), megalopa, and juvenile. Importantly, the prophenoloxidase was also present in the embryos, zoea, juvenile and adult crabs, but relatively lower in megalopa compared to those of other stages. Furthermore, the Sp-SPH mRNA expression showed a statistically significant increase (P<0.05) in both hemocyte and subcuticular epidermis at 24h, and in gill at 96h after challenge of V. parahaemolyticus determined by quantitative real-time PCR. Taken together, the live-bacterial-binding activity and the acute-phase response against bacterial infection of Sp-SPH suggested that it might function as an innate immune recognition molecule and play a key role in host defense against microbe invasion in the crab S. paramamosain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

PubMed Central

Gomis-Rüth, F X; Gómez, M; Bode, W; Huber, R; Avilés, F X

1995-01-01

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far. Images PMID:7556081

Isolation, characterization and antifungal activity of proteinase inhibitors from Capsicum chinense Jacq. Seeds.

PubMed

Dias, Germana Bueno; Gomes, Valdirene Moreira; Pereira, Umberto Zottich; Ribeiro, Suzanna F Ferreira; Carvalho, André O; Rodrigues, Rosana; Machado, Olga L Tavares; Fernandes, Kátia Valevski Sales; Ferreira, André Teixeira S; Perales, Jonas; Da Cunha, Maura

2013-01-01

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.

Action of plant proteinase inhibitors on enzymes of physiopathological importance.

PubMed

Oliva, Maria Luiza V; Sampaio, Misako U

2009-09-01

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

Gene polymorphisms of fibrinolytic enzymes in coal workers' pneumoconiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, L.C.; Tseng, J.C.; Hua, C.C.

2006-03-15

The authors assessed the gene polymorphisms of missense C/T polymorphism in exon 6 of the urokinase-plasminogen activator (PLAU) gene (PLAU P141L), A/u-repeat in intron 8 of the tissue-type plasminogen activator (PLAT) gene (PLAT TPA25 Alu insertion), and 4G/5G in the promoter region of the serine proteinase inhibitor, clade E (SERPINE) or plasminogen activator inhibitor type 1 gene (SERPINE1 -675 4G/5G) in 153 healthy volunteers and 154 retired coal miners with coal miners' pneumoconiosis (CWP). The CWP subjects included 94 individuals with simple pneumoconiosis and 60 individuals with progressive massive fibrosis presenting with worse pulmonary function. The distributions of genotypes ofmore » these three genes did not differ between the control and CWP subjects or between subjects with simple pneumoconiosis and those with progressive massive fibrosis. However, by assessing duration of work and its interaction with genotypes by means of logistic regression, the authors found the missense C/T polymorphism in exon 6 of the PLAU gene to be an effect modifier of the association between work duration and the development of progressive massive fibrosis.« less

Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries

PubMed Central

Glusa, Erika; Adam, Christine

2001-01-01

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2α (3 μM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC50=9.64±0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC50=9.23±0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 μM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC50=0.043 μg ml−1), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC50=0.14 μg ml−1) of the cathepsin G effect, whereas neither indomethacin (3 μM) nor the thrombin inhibitor hirudin (5 ATU ml−1) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC50=0.11 μg ml−1), heparin (IC50=0.48 μg ml−1) and suramin (IC50=1.85 μg ml−1) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium-dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation. PMID:11375259

Constitutive and regulated secretion of secretory leukocyte proteinase inhibitor by human intestinal epithelial cells.

PubMed

Si-Tahar, M; Merlin, D; Sitaraman, S; Madara, J L

2000-06-01

Epithelial cells participate in immune regulation and mucosal integrity by generating a range of biologically active mediators. In the intestine, little is known about the potential endogenous anti-inflammatory molecules. Secretory leukocyte proteinase inhibitor (SLPI) is a major serine proteinase inhibitor, a potent antibiotic, and thus a potential anti-inflammatory molecule, although it is not known if it is secreted by intestinal epithelial cells. We show, by reverse-transcription polymerase chain reaction, the presence of SLPI messenger RNA in human model intestinal epithelial cell lines (Caco2-BBE, T84, and HT29-Cl.19A) and human jejunum and colon biopsy specimens. The polymerase chain reaction product was cloned and sequenced and is identical to that of SLPI isolated previously from the human parotid gland. As analyzed by enzyme-linked immunosorbent assay, the constitutive secretion of SLPI occurs in a markedly polarized manner toward the apical surface and is enhanced by inflammatory mediators including tumor necrosis factor alpha and interleukin 1beta (approximately 3.5-fold increase over control value). SLPI release is also stimulated by activation of protein kinase C isoenzymes, but not by activation of adenosine 3',5'-cyclic monophosphate- or Ca(2+)-regulated signaling molecules. SLPI protein is detectable in intestinal lavage fluids collected from normal adult humans. Recombinant SLPI attenuates digestive enzyme (trypsin)- or leukocyte proteinase (elastase)-induced permeability alteration of a model epithelia in a dose-dependent manner. Moreover, SLPI exhibits an antibacterial activity against at least one major intestinal pathogen, Salmonella typhimurium. In contrast, SLPI does not influence epithelial barrier integrity as assessed by transepithelial conductance measurements or electrogenic ion transport. These results establish that human intestinal epithelium expresses and apically secretes SLPI, a molecule that may significantly contribute to the protection against attack from inflammatory cells and digestive enzymes, as well as against microbial infection.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells

PubMed Central

Peters, Haley L.; Tripathi, Satyendra C.; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R.; St. John, Lisa S.; Federico, Lorenzo; Meraz, Ismail M.; Roth, Jack A.; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L.; Heymach, John V.; Swisher, Stephen G.; Bernantchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J.

2017-01-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these re-invigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAM were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTL from healthy donors that had been expanded against select peptides. Finally, CTL specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. PMID:28254787

Genetic Analysis of Japanese Children With Acute Recurrent and Chronic Pancreatitis.

PubMed

Saito, Nobutomo; Suzuki, Mitsuyoshi; Sakurai, Yumiko; Nakano, Satoshi; Naritaka, Nakayuki; Minowa, Kei; Sai, Jin K; Shimizu, Toshiaki

2016-10-01

Causes of acute recurrent pancreatitis (ARP) or chronic pancreatitis (CP) are sometimes difficult to determine in children. In such patients, genetic analysis may prove helpful. The present study analyzed mutations of cationic trypsinogen (PRSS1), serine protease inhibitor Kazal type 1 (SPINK1), chymotrypsin C (CTRC), and carboxypeptidase A1 (CPA1) and investigated the clinical features of children with these mutations. Genetic analyses of mutations in these 4 genes were conducted in 128 patients with ARP or CP. Characteristics of the patients showing mutations were investigated using medical records. Fifty of the 128 (39.1%) subjects had at least 1 mutation (median age at onset, 7.6 years). Abdominal pain was the presenting symptom of pancreatitis in 48 of the 50 patients (96%). Fifteen of those 50 patients (30.0%) had a family history of pancreatitis. Gene mutations were present in PRSS1 in 26 patients, SPINK1 in 23, CTRC in 3, and CPA1 in 5. In the 31 patients with mutations in SPINK1, CTRC, or CPA1, 16 (51.6%) had homozygous or heterozygous mutations with other mutations. Three patients underwent surgery and another 4 patients underwent endoscopy to manage ARP or CP. Although 3 of the 7 patients complained of mild abdominal pain, none of those 7 patients experienced any obvious episode of ARP after treatment. In pediatric patients with idiopathic ARP and CP, genetic analysis is useful for identifying the cause of pancreatitis. Early endoscopic or surgical treatment prevents ARP by extending the interval between episodes of pancreatitis in this population.

Hereditary chronic pancreatitis

PubMed Central

Rosendahl, Jonas; Bödeker, Hans; Mössner, Joachim; Teich, Niels

2007-01-01

Hereditary chronic pancreatitis (HCP) is a very rare form of early onset chronic pancreatitis. With the exception of the young age at diagnosis and a slower progression, the clinical course, morphological features and laboratory findings of HCP do not differ from those of patients with alcoholic chronic pancreatitis. As well, diagnostic criteria and treatment of HCP resemble that of chronic pancreatitis of other causes. The clinical presentation is highly variable and includes chronic abdominal pain, impairment of endocrine and exocrine pancreatic function, nausea and vomiting, maldigestion, diabetes, pseudocysts, bile duct and duodenal obstruction, and rarely pancreatic cancer. Fortunately, most patients have a mild disease. Mutations in the PRSS1 gene, encoding cationic trypsinogen, play a causative role in chronic pancreatitis. It has been shown that the PRSS1 mutations increase autocatalytic conversion of trypsinogen to active trypsin, and thus probably cause premature, intrapancreatic trypsinogen activation disturbing the intrapancreatic balance of proteases and their inhibitors. Other genes, such as the anionic trypsinogen (PRSS2), the serine protease inhibitor, Kazal type 1 (SPINK1) and the cystic fibrosis transmembrane conductance regulator (CFTR) have been found to be associated with chronic pancreatitis (idiopathic and hereditary) as well. Genetic testing should only be performed in carefully selected patients by direct DNA sequencing and antenatal diagnosis should not be encouraged. Treatment focuses on enzyme and nutritional supplementation, pain management, pancreatic diabetes, and local organ complications, such as pseudocysts, bile duct or duodenal obstruction. The disease course and prognosis of patients with HCP is unpredictable. Pancreatic cancer risk is elevated. Therefore, HCP patients should strongly avoid environmental risk factors for pancreatic cancer. PMID:17204147

Hereditary pancreatitis of 3 Chinese children

PubMed Central

Dai, Li-Na; Chen, Ying-Wei; Yan, Wei-Hui; Lu, Li-Na; Tao, Yi-Jing; Cai, Wei

2016-01-01

Abstract Background: Hereditary pancreatitis (HP) is quite rare and is distinguished by incomplete penetrance presentation as early-onset relapsing pancreatitis, usually beginning in childhood. HP is now known to be commonly relevant to mutations in the PRSS1 (gene-encoding cationic trypsinogen), SPINK1 (serine protease inhibitor, Kazal type 1), CFTR (cystic fibrosis), carboxypeptidase A1 (CPA1), and chymotrypsin C (CTRC) genes as reported in some Caucasian studies. HP has a variable spectrum of severity and may develop complications. Methods & Results: We describe the clinical course of 3 preschool children, hospitalized with postprandial abdominal pain, whose laboratory tests showed high serum amylase. Similar episodes of abdominal pain led to readmission, and the patients recovered quickly after using symptomatic therapy. The condition of the first boy, who developed a pancreatic tail pseudocyst and splenic infarction, was especially complicated. The boy underwent 2 endoscopic retrograde cholangiopancreatographies and stenting, along with a surgical procedure that completely relieved his symptoms for 3 months. The 3 patients and their parents were given genetic testing. All of the patients carried 1 or more gene mutations inherited from their mothers, fathers, or both parents; however, none of the parents were affected. Conclusion: For children with repeated pancreatitis, clinicians should consider HP in the differential diagnosis. It is reliable to perform gene sequencing on suspicious patients and their parents. Multidisciplinary and comprehensive treatment should be recommended to manage HP and its complications. Cholangiopancreatography and stenting is a relatively minimally invasive approach when compared with surgery and can be tried as an early intervention. Surgical procedures should be reserved for patients with complications. PMID:27603351

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

PubMed Central

Duan, Jun; Wang, Genhong; Wang, Lingyan; Li, Youshan; Xiang, Zhonghuai; Xia, Qingyou

2012-01-01

In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein. PMID:22348050

Interaction between tumor cell surface receptor RAGE and proteinase 3 mediates prostate cancer metastasis to bone

PubMed Central

Kolonin, Mikhail G.; Sergeeva, Anna; Staquicini, Daniela I.; Smith, Tracey L.; Tarleton, Christy A.; Molldrem, Jeffrey J.; Sidman, Richard L.; Marchiò, Serena; Pasqualini, Renata; Arap, Wadih

2017-01-01

Human prostate cancer often metastasizes to bone, but the biological basis for such site-specific tropism remains largely unresolved. Recent work led us to hypothesize that this tropism may reflect pathogenic interactions between RAGE, a cell surface receptor expressed on malignant cells in advanced prostate cancer, and proteinase 3 (PR3), a serine protease present in inflammatory neutrophils and hematopoietic cells within the bone marrow microenvironment. In this study, we establish that RAGE-PR3 interaction mediates homing of prostate cancer cells to the bone marrow. PR3 bound to RAGE on the surface of prostate cancer cells in vitro, inducing tumor cell motility through a non-proteolytic signal transduction cascade involving activation and phosphorylation of ERK1/2 and JNK1. In preclinical models of experimental metastasis, ectopic expression of RAGE on human prostate cancer cells was sufficient to promote bone marrow homing within a short time frame. Our findings demonstrate how RAGE-PR3 interactions between human prostate cancer cells and the bone marrow microenvironment mediate bone metastasis during prostate cancer progression, with potential implications for prognosis and therapeutic intervention. PMID:28428279

Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

NASA Astrophysics Data System (ADS)

Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice.

PubMed

Almeida-Reis, Rafael; Theodoro-Junior, Osmar A; Oliveira, Bruno T M; Oliva, Leandro V; Toledo-Arruda, Alessandra C; Bonturi, Camila R; Brito, Marlon V; Lopes, Fernanda D T Q S; Prado, Carla M; Florencio, Ariana C; Martins, Mílton A; Owen, Caroline A; Leick, Edna A; Oliva, Maria L V; Tibério, Iolanda F L C

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL / 6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNF α -, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2 α , collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNF α -positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

PubMed Central

Theodoro-Junior, Osmar A.; Oliveira, Bruno T. M.; Oliva, Leandro V.; Toledo-Arruda, Alessandra C.; Bonturi, Camila R.; Brito, Marlon V.; Prado, Carla M.; Florencio, Ariana C.; Martins, Mílton A.; Owen, Caroline A.; Oliva, Maria L. V.

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL/6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNFα-, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2α, collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNFα-positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease. PMID:28466019

The serine proteinase inhibitory proteins of the chondrodystrophoid (beagle) and non-chondrodystrophoid (greyhound) canine intervertebral disc.

PubMed

Melrose, J; Taylor, T K; Ghosh, P

1997-06-01

Trypsin inhibitory proteins of low buoyant density (p < or = 1.35 g/mL) fractions were prepared by CsCl density gradient ultracentrifugation of 4 M guanidinium hydrochloride extracts of lumbar beagle and greyhound annulus fibrosus and nucleus pulposus from animals aged 1 to 6 years. Affinity blotting with biotinylated trypsin was used to identify active trypsin inhibitory protein species; these species were also identified immunologically by Western blotting using antibodies against bovine pancreatic trypsin inhibitor (BPTI), and human inter-alpha-trypsin inhibitor (ITI). None of the trypsin inhibitory species evident in Western blots were reactive with anti-human alpha1-proteinase inhibitor (alpha-1-PI), alpha2-macroglobulin or secretory leucocyte proteinase inhibitor. The greyhound intervertebral disc samples generally had higher levels of active trypsin inhibitor species per unit weight of tissue extracted, and a more extensive range of inhibitor species. Inhibitor species of 30, 32, 34 kDa were identified in both beagle and greyhound intervertebral disc samples; these species were generally most prominent in the annulus fibrosus samples. In contrast, the nucleus pulposus samples contained relatively large trypsin inhibitor species; the anti-BPTI detected an inhibitor species of approximately 85-90 kDa; anti-ITI detected species of 120-250 kDa; biotinylated trypsin detected species of 60-110 kDa. A small molecular mass trypsin inhibitor species of 6 kDa, which was of similar mobility to BPTI, was also detected in annulus fibrosus samples; however, this species did not react with anti-BPTI.

Wheat Subtilisin/Chymotrypsin Inhibitor (WSCI) as a scaffold for novel serine protease inhibitors with a given specificity.

PubMed

Tedeschi, Francesca; Di Maro, Antimo; Facchiano, Angelo; Costantini, Susan; Chambery, Angela; Bruni, Natalia; Capuzzi, Valeria; Ficca, Anna Grazia; Poerio, Elia

2012-10-30

WSCI (Wheat Subtilisin/Chymotrypsin Inhibitor) is a small protein belonging to the Potato inhibitor I family exhibiting a high content of essential amino acid. In addition to bacterial subtilisins and mammalian chymotrypsins, WSCI inhibits chymotrypsin-like activities isolated from digestive traits of a number of insect larvae. In vivo, as suggested for many plant proteinase inhibitors, WSCI seems to play a role of natural defence against attacks of pests and pathogens. The functional region of WSCI, containing the inhibitor reactive site (Met48-Glu49), corresponds to an extended flexible loop (Val42-Asp53) whose architecture is somehow stabilized by a number of secondary interactions established with a small β-sheet located underneath. The aim of this study was to employ a WSCI molecule as a stable scaffold to obtain recombinant inhibitors with new acquired anti-proteinase activity or, alternatively, inactive WSCI variants. A gene sequence coding for the native WSCI, along with genes coding for muteins with different specficities, could be exploited to obtain transformed non-food use plants with improved insect resistance. On the other hand, the genetic transformation of cereal plants over-expressing inactive WSCI muteins could represent a possible strategy to improve the nutritional quality of cereal-based foods, without risk of interference with human or animal digestive enzymes. Here, we described the characterization of four muteins containing single/multiple amino acid substitutions at the WSCI reactive site and/or at its proximity. Modalities of interaction of these muteins with proteinases (subtilisin, trypsin and chymotrypsin) were investigated by time course hydrolysis and molecular simulations studies.

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

PubMed

O'Hara, M B; Hageman, J H

1990-08-01

Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.

Circular dichroism of stem bromelain: a third spectral class within the family of cysteine proteinases.

PubMed Central

Arroyo-Reyna, A; Hernandez-Arana, A; Arreguin-Espinosa, R

1994-01-01

Two forms of stem bromelain (EC 3.4.22.4) were isolated from commercial, crude and chromatographically purified preparations of the enzyme by means of gel-filtration and cation-exchange liquid chromatography. These forms possess nearly identical secondary and tertiary structures, as judged from their circular dichroism (c.d.) spectra. The spectral characteristics of stem bromelain suggest that this enzyme belongs to the alpha + beta protein class, as other cysteine proteinases do. In agreement with these results, quantitative estimation of secondary structures yielded amounts similar to those for papain and proteinase omega. However, the bromelain c.d. curve is clearly distinguishable from those reported for papain and proteinase omega, on one hand, and that of chymopapain, on the other. Thus, it is apparent that there are at least three types of c.d. spectra associated with the family of cysteine proteinases. PMID:8198520

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

PubMed

Peters, Haley L; Tripathi, Satyendra C; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R; St John, Lisa S; Federico, Lorenzo; Meraz, Ismail M; Roth, Jack A; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L; Heymach, John V; Swisher, Stephen G; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J

2017-04-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR . ©2017 American Association for Cancer Research.

The action of neutrophil serine proteases on elastin and its precursor.

PubMed

Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

2012-01-01

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting.

PubMed

Fernández Gimenez, A V; García-Carreño, F L; Navarrete del Toro, M A; Fenucci, J L

2001-10-01

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

PubMed Central

He, Shaoheng; Walls, Andrew F

1998-01-01

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals.Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site.Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils, 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7×10−13 mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h.The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme.Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum.Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation. PMID:9884078

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

PubMed

Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

1995-10-06

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

PubMed

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

PubMed

Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

2012-09-01

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

PubMed

van de Locht, A; Lamba, D; Bauer, M; Huber, R; Friedrich, T; Kröger, B; Höffken, W; Bode, W

1995-11-01

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite.

Purification of cynarases from artichoke (Cynara scolymus L.): enzymatic properties of cynarase A.

PubMed

Sidrach, Lara; García-Cánovas, Francisco; Tudela, José; Rodríguez-López, José Neptuno

2005-01-01

Aspartic proteinases from flowers of Cynara cardunculus have been extensively studied and long used as coagulants in the manufacture of several traditional Spanish and Portuguese cheeses. These endopeptidases are called cardosins or cynarases, depending on the authors. However, the proteinases of another plant of the genus Cynara, the artichoke (Cynara scolymus), are less known, probably because the flower of this plant is usually consumed as a vegetable. In the study described here, three proteinases (cynarases A, B and C) with milk-clotting properties were purified from the stigma of artichoke. All three proteinases are glycoproteins and composed of a one large and one small subunit. The enzymatic properties of cynarase A, a glycoprotein containing N-linked high mannose type glycans, which express maximum activity at pH 5.0 and 70 degrees C, were studied in detail. Catalytic and inhibition studies indicated that this cynarase is of the aspartic acid type. The results indicate artichoke extract could also be used in the milk industry in the same way as the extract obtained from the flower of C. cardunculus.

Amyloid-degrading ability of nattokinase from Bacillus subtilis natto.

PubMed

Hsu, Ruei-Lin; Lee, Kung-Ta; Wang, Jung-Hao; Lee, Lily Y-L; Chen, Rita P-Y

2009-01-28

More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

Inactivation of Human Neutrophil Elastase by 1, 2, 5 – Thiadiazolidin-3-one 1, 1 Dioxide-based Sulfonamides

PubMed Central

Li, Yi; Yang, Qingliang; Dou, Dengfeng; Alliston, Kevin R.; Groutas, William C.

2008-01-01

The interaction of a series of 1, 2, 5 –thiadiazolidin-3-one 1, 1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S′ subsites, had a profound effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elastase (HNE) and was devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results of these studies demonstrate that exploitation of differences in the S′ subsites of HNE and PR 3 can lead to highly selective inhibitors of HNE. PMID:17976994

SNAKE VENOMICS OF Crotalus tigris: THE MINIMALIST TOXIN ARSENAL OF THE DEADLIEST NEARTIC RATTLESNAKE VENOM

PubMed Central

CALVETE, Juan J.; PÉREZ, Alicia; LOMONTE, Bruno; SÁNCHEZ, Elda E.; SANZ, Libia

2012-01-01

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7–8 gene products from 6 toxin families: the presynaptic β-neurotoxic heterodimeric PLA2, Mojave toxin, and two serine proteinases comprise, respectively, 66% and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1–2 PIII-SVMPs, each represents 0.1–5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend towards neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by paedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, C. horridus, C. oreganus helleri, C. scutulatus scutulatus, and S. catenatus catenatus, indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South American and North American Crotalus. PMID:22181673

Proteinase activity of prevotella species associated with oral purulent infection.

PubMed

Yanagisawa, Maki; Kuriyama, Tomoari; Williams, David W; Nakagawa, Kiyomasa; Karasawa, Tadahiro

2006-05-01

Prevotella intermedia and Prevotella nigrescens are often regarded as principal causes of acute dentoalveolar infection; however, other species within the genus are also known to be associated with such infection. The aim of this study was to determine the in vitro proteolytic activity of these different Prevotella species that have been implicated with dentoalveolar infection. A total of 234 strains were obtained from pus specimens from dentoalveolar infection and from the plaque of healthy volunteers. Prevotella loescheii, Prevotella oralis, Prevotella melaninogenica, Prevotella buccae, and Prevotella denticola were all shown to have a proteolytic activity (8.5-10.5 x 10(-8) A-units) lower than that of P. intermedia and P. nigrescens (21.1-23.5 x 10(-8) A-units). In the case of P. loescheii, P. melaninogenica, and P. intermedia, the level of proteolytic activity for clinical strains was significantly (P < 0.05) higher than that recorded for commensal strains. Proteolytic activity for all species of Prevotella examined was inhibited by N-ethylmaleimide and phenymethylsulfonyl fluoride. This study suggests that Prevotella species associated with oral purulent infection produce cysteine and serine proteinases and that in certain species of Prevotella, the strains involved in infection exhibit higher proteolytic activity when compared with strains from healthy sites.

High prevalence of the c.74A>C SPINK1 variant in miniature and standard Schnauzers.

PubMed

Furrow, E; Armstrong, P J; Patterson, E E

2012-01-01

Variants in the serine protease inhibitor Kazal type 1 (SPINK1) gene have been associated with pancreatitis in Miniature Schnauzers. Replication of the association in an independent population is necessary to determine if genetic screening for SPINK1 variants should be considered in clinical practice. An association between the SPINK1 exonic variant c.74A > C and pancreatitis exists in Miniature Schnauzers. In addition, the variant is absent or rare in Standard Schnauzers, a related breed that is not reported to have an increased risk for pancreatitis. Case-control study. Seventeen Miniature Schnauzers with pancreatitis (cases), 60 mature Miniature Schnauzers with no substantial history of gastrointestinal signs in their lifetime (controls), and 31 Standard Schnauzers of unknown pancreatitis status. A PCR-RFLP assay was used to genotype dogs for the c.74A > C SPINK1 variant. Allele and genotype frequencies were reported for Schnauzers and compared between case and control Miniature Schnauzers. The c.74A > C variant was the major allele in both Schnauzer breeds with a frequency of 0.77 in Miniatures and 0.55 in Standards. The allele and genotype frequencies were similar between Miniature Schnauzers with and without a history of pancreatitis and did not impart an increased risk for pancreatitis. Genotyping a larger population of the Miniature Schnauzer breed than a previous study, along with a Standard Schnauzer cohort, demonstrated that the SPINK1 c.74A > C variant is a common polymorphism in the Schnauzer lineage. Furthermore, we were unable to confirm a relationship between the variant and clinically detectable pancreatitis in Miniature Schnauzers. Copyright © 2012 by the American College of Veterinary Internal Medicine.

Functional significance of SPINK1 promoter variants in chronic pancreatitis.

PubMed

Derikx, Monique H M; Geisz, Andrea; Kereszturi, Éva; Sahin-Tóth, Miklós

2015-05-01

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation. Copyright © 2015 the American Physiological Society.

Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss.

PubMed

Krieg, S A; Fan, X; Hong, Y; Sang, Q-X; Giaccia, A; Westphal, L M; Lathi, R B; Krieg, A J; Nayak, N R

2012-09-01

Recurrent pregnancy loss (RPL) occurs in ∼5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

PubMed

Sharma, S; Tyagi, R; Gupta, M N; Singh, T P

2001-01-01

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.

Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella: comparison of midgut proteinases from susceptible and resistant larvae.

PubMed

Johnson, D E; Brookhart, G L; Kramer, K J; Barnett, B D; McGaughey, W H

1990-03-01

Midgut homogenates from susceptible and resistant strains of the Indian meal moth, Plodia interpunctella, were compared for their ability to activate the entomocidal parasporal crystal protein from Bacillus thuringiensis. The properties of midgut proteinases from both types of larvae were also examined. Electrophoretic patterns of crystal protein from B. thuringiensis subspecies kurstaki (HD-1) and aizawai (HD-133 and HD-144) were virtually unchanged following digestion by either type of midgut homogenate. Changes in pH (9.5 to 11.5) or midgut homogenate concentration during digestion failed to substantially alter protein electrophoretic patterns of B. thuringiensis HD-1 crystal toxin. In vitro toxicity of crystal protein activated by either type of midgut preparation was equal toward cultured insect cells from either Manduca sexta or Choristoneura fumiferana. Electrophoresis of midgut extracts in polyacrylamide gels containing gelatin as substrate also yielded matching mobility patterns of proteinases from both types of midguts. Quantitation of midgut proteolytic activity using tritiated casein as a substrate revealed variation between midgut preparations, but no statistically significant differences between proteolytic activities from susceptible and resistant Indian meal moth larvae. Inhibition studies indicated that a trypsin-like proteinase with maximal activity at pH 10 is a major constituent of Indian meal moth midguts. The results demonstrated that midguts from susceptible and resistant strains of P. interpunctella are similar both in their ability to activate B. thuringiensis protoxin and in their proteolytic activity.

Chymotryptic specificity determinants in the 1.0 Å structure of the zinc-inhibited human tissue kallikrein 7

PubMed Central

Debela, Mekdes; Hess, Petra; Magdolen, Viktor; Schechter, Norman M.; Steiner, Thomas; Huber, Robert; Bode, Wolfram; Goettig, Peter

2007-01-01

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70–80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn2+ and Cu2+ at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His99 close to the catalytic triad accounts for the noncompetitive Zn2+ inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition. PMID:17909180

Catalytic autoantibodies against myelin basic protein (MBP) isolated from serum of autistic children impair in vitro models of synaptic plasticity in rat hippocampus.

PubMed

Gonzalez-Gronow, Mario; Cuchacovich, Miguel; Francos, Rina; Cuchacovich, Stephanie; Blanco, Angel; Sandoval, Rodrigo; Gomez, Cristian Farias; Valenzuela, Javier A; Ray, Rupa; Pizzo, Salvatore V

2015-10-15

Autoantibodies from autistic spectrum disorder (ASD) patients react with multiple proteins expressed in the brain. One such autoantibody targets myelin basic protein (MBP). ASD patients have autoantibodies to MBP of both the IgG and IgA classes in high titers, but no autoantibodies of the IgM class. IgA autoantibodies act as serine proteinases and degrade MBP in vitro. They also induce a decrease in long-term potentiation in the hippocampi of rats either perfused with or previously inoculated with this IgA. Because this class of autoantibody causes myelin sheath destruction in multiple sclerosis (MS), we hypothesized a similar pathological role for them in ASD. Copyright © 2015 Elsevier B.V. All rights reserved.

Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

PubMed Central

Padua, Maria B; Hansen, Peter J

2008-01-01

Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth. PMID:18218135

Kallistatin as a marker of microvascular complications in children and adolescents with type 1 diabetes mellitus: Relation to carotid intima media thickness.

PubMed

El-Asrar, Mohamed A; Andrawes, Nevine G; Ismail, Eman A; Salem, Shaimaa Mh

2015-12-01

In diabetes, angiogenesis is disturbed, contributing to proliferative retinopathy, nephropathy and neuropathy. Kallistatin, a serine proteinase inhibitor, has anti-angiogenic effects. We assessed serum kallistatin in children and adolescents with type 1 diabetes as a potential marker for microvascular complications and its relation to carotid intima media thickness (CIMT). Sixty patients with type 1 diabetes were divided into two groups according to the presence of microvascular complications and compared with 30 healthy controls. High-sensitivity C-reactive protein (hs-CRP), HbA1c, urinary albumin creatinine ratio (UACR), kallistatin levels and CIMT were assessed. Kallistatin levels were significantly higher in patients with microvascular complications (9.9 ± 2.38 ng/mL) and those without complications (5.0 ± 1.5 ng/mL) than in healthy controls (1.39 ± 0.55 ng/mL; p<0.001). Kallistatin was increased in patients with microalbuminuria compared with the normoalbuminuric group (p<0.001). Positive correlations were found between kallistatin and disease duration, fasting blood glucose, HbA1c, triglycerides, total cholesterol, hs-CRP, UACR and CIMT (p<0.05). A kallistatin cut-off value at 6.1 ng/mL could differentiate patients with and without microvascular complications, with a sensitivity of 96.87% and specificity of 93.75%. Increased kallistatin levels in type 1 diabetes and its relation with CIMT may reflect vascular dysfunction and suggest a link between micro- and macro-angiopathy. © The Author(s) 2015.

Neutrophil proteinase 3 (PR3) acts on protease-activated receptor-2 (PAR-2) to enhance vascular endothelial cell barrier function

PubMed Central

Kuckleburg, Christopher J.; Newman, Peter J.

2013-01-01

The principle role of the vascular endothelium is to present a semi-impermeable barrier to soluble factors and circulating cells, while still permitting the passage of leukocytes from the bloodstream into the tissue. The process of diapedesis involves the selective disruption of endothelial cell junctions, an event that could in theory compromise vascular integrity. It is therefore somewhat surprising that neutrophil transmigration does not significantly impair endothelial barrier function. We examined whether neutrophils might secrete factors that promote vascular integrity during the latter stages of neutrophil transmigration, and found that neutrophil proteinase 3 (PR3) – a serine protease harbored in azurophilic granules – markedly enhanced barrier function in endothelial cells. PR3 functioned in this capacity both in its soluble form and in a complex with cell-surface NB1. PR3-mediated enhancement of endothelial cell junctional integrity required its proteolytic activity, as well as endothelial cell expression of the protease-activated receptor, PAR-2. Importantly, PR3 suppressed the vascular permeability changes and disruption of junctional proteins induced by the action of PAR-1 agonists. These findings establish the potential for neutrophil-derived PR3 to play a role in reestablishing vascular integrity following leukocyte transmigration, and in protecting endothelial cells from PAR-1-induced permeability changes that occur during thrombotic and inflammatory events. PMID:23202369

Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

PubMed

Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

2005-07-01

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

PubMed

Gupta, M N; Tyagi, R; Sharma, S; Karthikeyan, S; Singh, T P

2000-05-15

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

The vagina of women infected with Trichomonas vaginalis has numerous proteinases and antibody to trichomonad proteinases.

PubMed

Alderete, J F; Newton, E; Dennis, C; Neale, K A

1991-12-01

Patients with trichomoniasis have serum antibody to numerous T. vaginalis cysteine proteinases, indicating that the proteinases are expressed in vivo. It was important, therefore, to examine for the presence of soluble trichomonad proteinases and/or antibody to the proteinases in the vagina of infected women. Vaginal washes (VWs) from 20 women were examined for the presence of proteinases by electrophoresis using acrylamide co-polymerised with gelatin as the indicator system. Antibody to proteinases in VWs was detected by an immunoprecipitation assay involving protein A-bearing Staphylococcus aureus first coated with anti-human immunoglobulin G (IgG) antibody, which was then added to VWs. For VWs having soluble proteinases, the bacteria were used to determine whether immune complexes between antibody and proteinases were present. VWs without soluble proteinases were incubated with the anti-human IgG treated bacteria before adding to detergent extracts of T. vaginalis. Individual isolates from the patients examined in this study were also analysed by one- and two-dimensional electrophoresis for their proteinase content. Finally, VWs were from patients without any history of other sexually transmitted diseases (STDs) as well as from individuals having numerous other STDs, including yeast, group B streptococcus, chlamydia, and syphilis. Approximately one-third of patients had soluble proteinases in the VWs; the remaining two-thirds (70%) of patients and normal women had no detectable proteinases in VWs. Half of the patients without soluble proteinases had IgG which, when bound to S. aureus, immunoprecipitated many proteinases from a detergent extract of T. vaginalis. All soluble proteinases and those precipitated from trichomonal extracts were inhibited by inhibitors of cysteine proteinases. Finally, patients having trichomoniasis in addition to numerous other STD agents, including yeast, group B streptococcus, chlamydia, and syphilis did not have soluble proteinases in VWs. Equally noteworthy, some patients with soluble proteinases in VWs did not have other detectable STD agents. Proteinases were detected in the vagina of some patients with trichomoniasis, and in most cases the proteinases were complexed with IgG, which was precipitated by S. aureus. Patients without soluble proteinases in VWs also had antibody specifically to trichomonad proteinases, again demonstrating both the expression and immunogenic nature of the proteinases in vivo. The absence of soluble proteinases in normal women and in patients having other STD agents as well as the presence of proteinases in VWs of patients without other detectable STD pathogens reinforced the idea that the proteinases were of T. vaginalis parasite origin. The findings of this study indicate that proteinases may be important to the T. vaginalis-host interrelationship.

Comparative venomics of the Prairie Rattlesnake (Crotalus viridis viridis) from Colorado: Identification of a novel pattern of ontogenetic changes in venom composition and assessment of the immunoreactivity of the commercial antivenom CroFab®.

PubMed

Saviola, Anthony J; Pla, Davinia; Sanz, Libia; Castoe, Todd A; Calvete, Juan J; Mackessy, Stephen P

2015-05-21

Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America. Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms. Copyright © 2015 Elsevier B.V. All rights reserved.

An atypical proprotein convertase in Giardia lamblia differentiation.

PubMed

Davids, B J; Gilbert, M A; Liu, Q; Reiner, D S; Smith, A J; Lauwaet, T; Lee, C; McArthur, A G; Gillin, F D

2011-02-01

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation. Copyright © 2010 Elsevier B.V. All rights reserved.

An Atypical Proprotein Convertase in Giardia lamblia Differentiation

PubMed Central

Davids, B. J.; Gilbert, M. A.; Liu, Q.; Reiner, D. S.; Smith, A. J.; Lauwaet, T.; Lee, C.; McArthur, A. G.; Gillin, F. D.

2010-01-01

Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologues distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1 mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation. PMID:21075147

DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

PubMed

Nguyen, Uyen T; Burrows, Lori L

2014-09-18

Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (<50% of control) to polystyrene. Treatment of established 72h biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with <25% biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

Venomic and antivenomic analyses of the Central American coral snake, Micrurus nigrocinctus (Elapidae).

PubMed

Fernández, Julián; Alape-Girón, Alberto; Angulo, Yamileth; Sanz, Libia; Gutiérrez, José María; Calvete, Juan J; Lomonte, Bruno

2011-04-01

The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a "venomics" approach. Nearly 50 venom peaks were resolved by RP-HPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A(2) (PLA(2)), three-finger toxin (3FTx), l-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA(2)s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain α-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA(2)s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs.

[A case of mixed connective tissue disease positive for proteinase 3 antineutrophil cytoplasmic antibody in a patient with slowly progressive type 1 diabetes mellitus and chronic thyroiditis].

PubMed

Michitsuji, Tohru; Horai, Yoshiro; Sako, Ayaka; Asano, Taro; Iwanaga, Nozomi; Izumi, Yasumori; Kawakami, Atsushi

2017-01-01

  A female in her sixties with slowly progressive type 1 diabetes mellitus (SPT1DM) and chronic thyroiditis was referred to our rheumatology department with swelling in her fingers. A prominent atherosclerotic lesion was revealed upon brain magnetic resonance imaging, and she was found to have mixed connective tissue disease (MCTD) positive for proteinase 3 (PR3)-antineutrophil cytoplasmic antibody (ANCA). This rare case of MCTD accompanying SPT1DM and PR3-ANCA suggested that a synergy between MCTD and PR3-ANCA triggers atherosclerosis.

Enhanced response of a proteinase K-based conductometric biosensor using nanoparticles.

PubMed

Nouira, Wided; Maaref, Abderrazak; Elaissari, Hamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

2014-07-23

Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 µs. These results are greater than that found without any nanoparticles (maximum response of 10 µs). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained.

Low-dose retinoic acid enhances in vitro invasiveness of human oral squamous-cell-carcinoma cell lines

PubMed Central

Uchida, D; Kawamata, H; Nakashiro, K; Omotehara, F; Hino, S; Hoque, M O; Begum, N-M; Yoshida, H; Sato, M; Fujimori, T

2001-01-01

Retinoids inhibit the proliferation of several types of tumour cells, and are used for patients with several malignant tumours. In this study, we examined the effect of retinoic acids (RAs) on the invasive potentials of the oral squamous cell carcinoma (SCC) cells, BHY and HNt. BHY cells expressed all of retinoid nuclear receptors (RARα, β, γ, and RXRα) and cytoplasmic retinoic acid binding proteins (CRABP1 and CRABP2). HNt cells lacked the expression of RARβ, but expressed other nuclear receptors and CRABPs. All-trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cisRA) (10−6and 10−7M) inhibited the growth of the cells, but low-dose ATRA and 13-cisRA (10−8M) marginally affected the growth of the cells. Surprisingly, low-dose RAs enhanced the activity of tissue-type plasminogen activator (tPA), and activated pro-matrix metalloproteinases (proMMP2 and proMMP9). Activation of proMMP2 and proMMP9 was inhibited by aprotinin, a serine-proteinase, tPA inhibitor. Furthermore, low-dose RAs enhanced the in vitro invasiveness of BHY cells. These results indicate that low-dose RAs enhances the in vitro invasiveness of oral SCC cells via an activation of proMMP2 and proMMP9 probably mediated by the induction of tPA. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11437413

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation

PubMed Central

Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

2015-01-01

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. PMID:25495009

Immediate-type allergic and protease-mediated reactions are involved in scratching behaviour induced by topical application of Dermatophagoides farinae extract in NC/Nga mice.

PubMed

Yamada, Yoshihito; Ueda, Yuhki; Nakamura, Aki; Kanayama, Shoji; Tamura, Rie; Hashimoto, Kei; Matsumoto, Tatsumi; Ishii, Ritsuko

2018-04-01

Atopic dermatitis (AD)-like dermatitis can be induced by repeated topical application of an ointment containing Dermatophagoides farinae body (Dfb) extract in NC/Nga mice. This AD-like murine model also exhibits a biphasic increase in the number of scratching behaviour after topical application of Dfb ointment. In this study, we investigated the possible mechanisms underlying the scratching behaviour in each phase. An increase in the content of mast cell-derived mediators such as histamine and 5-hydroxytryptamine in the lesional skin and increased vascular permeability were observed in the early phase after the Dfb ointment application. Chlorpheniramine (H 1 receptor antagonist) and cromoglycate (mast cell stabilizer) reduced the scratching behaviour in the early phase but not that in the later phase. Furthermore, the content of various endogenous pruritogens such as interleukin-31 and thymic stromal lymphopoietin in the lesional skin was increased 1 or 24 hours after the Dfb ointment application. Elevated expression of proteinase-activated receptor-2 (PAR-2) was also observed in the epidermis. Finally, gabexate (serine protease inhibitor) reduced the scratching behaviour in both phases, and anti-PAR2 antibody also showed a tendency to reduce both scratching behaviours. These findings suggest that immediate-type allergic reactions caused by mast cell degranulation and PAR-2 activation by proteases are involved in the scratching behaviour in this AD-like model. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.

α 1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based onmore » biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography. PLOS« less

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, C.W.; Mangel, W.F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, Carl W.; Mangel, Walter F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Co-factor activated recombinant adenovirus proteinases

DOEpatents

Anderson, Carl W.; Mangel, Walter F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Co-factor activated recombinant adenovirus proteinases

DOEpatents

Anderson, C.W.; Mangel, W.F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

PubMed

Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

2010-06-01

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Drosophila serpin 4 functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases.

PubMed

Osterwalder, Thomas; Kuhnen, Angela; Leiserson, William M; Kim, You-Seung; Keshishian, Haig

2004-06-16

The proteolytic processing of neuropeptide precursors is believed to be regulated by serine proteinase inhibitors, or serpins. Here we describe the molecular cloning and functional expression of a novel member of the serpin family, Serine protease inhibitor 4 (Spn4), that we propose is involved in the regulation of peptide maturation in Drosophila. The Spn4 gene encodes at least two different serpin proteins, generated by alternate splicing of the last coding exon. The closest vertebrate homolog to Spn4 is neuroserpin. Like neuroserpin, one of the Spn4 proteins (Spn4.1) features a unique C-terminal extension, reminiscent of an endoplasmic reticulum (ER) retention signal; however, Spn4.1 and neuroserpin have divergent reactive site loops, with Spn4.1 showing a generic recognition site for furin/SPC1, the founding member of the intracellularly active family of subtilisin-like proprotein convertases (SPCs). In vitro, Spn4.1 forms SDS-stable complexes with the SPC furin and directly inhibits it. When Spn4.1 is overexpressed in specific peptidergic cells of Drosophila larvae, the animals exhibit a phenotype consistent with disrupted neuropeptide processing. This observation, together with the unique combination of an ER-retention signal, a target sequence for SPCs in the reactive site loop, and the in vitro inhibitory activity against furin, strongly suggests that Spn4.1 is an intracellular regulator of SPCs.

Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs)

PubMed Central

Breyer, Johanna; Wemheuer, Wiebke M.; Wrede, Arne; Graham, Catherine; Benestad, Sylvie L.; Brenig, Bertram; Richt, Jürgen A.; Schulz-Schaeffer, Walter J.

2012-01-01

Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrPSc). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrPSc against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorbtion assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrPSc after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrPSc can be very different. The results obtained here may be helpful during the development or improvement of a PrPSc detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrPSc that may stabilize the aggregates. PMID:22226540

Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon-Chung, K.J.; Wickes, B.L.; Merz, W.G.

1988-07-01

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infectionmore » in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates.« less

Peptide fingerprinting of the sea anemone Heteractis magnifica mucus revealed neurotoxins, Kunitz-type proteinase inhibitors and a new β-defensin α-amylase inhibitor.

PubMed

Sintsova, Oksana; Gladkikh, Irina; Chausova, Victoria; Monastyrnaya, Margarita; Anastyuk, Stanislav; Chernikov, Oleg; Yurchenko, Ekaterina; Aminin, Dmitriy; Isaeva, Marina; Leychenko, Elena; Kozlovskaya, Emma

2018-02-20

Sea anemone mucus, due to its multiple and vital functions, is a valuable substance for investigation of new biologically active peptides. In this work, compounds of Heteractis magnifica mucus were separated by multistage liquid chromatography and resulting fractions were analyzed by MALDI-TOF MS. Peptide maps constructed according to the molecular masses and hydrophobicity showed presence of 326 both new and known peptides. Several major peptides from mucus were identified, including the sodium channel toxin RpII isolated earlier from H. magnifica, and four Kunitz-type proteinase inhibitors identical to H. crispa ones. Kunitz-type transcript diversity was studied and sequences of mature peptides were deduced. New β-defensin α-amylase inhibitor, a homolog of helianthamide from Stichodactyla helianthus, was isolated and structurally characterized. Overall, H. magnifica is a source of biologically active peptides with great pharmacological potential. Proteinase and α-amylase inhibitors along with toxins are major components of H. magnifica mucus which play an important role in the successful existence of sea anemones. Obtained peptide maps create a basis for more accurate identification of peptides during future transcriptomic/genomic studies of sea anemone H. magnifica. Copyright © 2017 Elsevier B.V. All rights reserved.

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

DOE PAGES

Stojanoski, Vlatko; Adamski, Carolyn J.; Hu, Liya; ...

2016-04-12

Serine β-lactamases are bacterial enzymes that hydrolyze β- lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. Here, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M- 14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutantmore » were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. In addition, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. Our findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.« less

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

PubMed Central

Stojanoski, Vlatko; Adamski, Carolyn J.; Hu, Liya; Mehta, Shrenik C.; Sankaran, Banumathi; Zwart, Peter; Prasad, B.V. Venkataram; Palzkill, Timothy

2016-01-01

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine by glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48 and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intra-helical hydrogen bond network compared to the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability. PMID:27073009

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stojanoski, Vlatko; Adamski, Carolyn J.; Hu, Liya

Serine β-lactamases are bacterial enzymes that hydrolyze β- lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. Here, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M- 14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutantmore » were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. In addition, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. Our findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.« less

Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

PubMed

Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

2016-10-01

Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Phosphoserine Phosphatase Is Required for Serine and One-Carbon Unit Synthesis in Hydrogenobacter thermophilus.

PubMed

Kim, Keugtae; Chiba, Yoko; Kobayashi, Azusa; Arai, Hiroyuki; Ishii, Masaharu

2017-11-01

Hydrogenobacter thermophilus is an obligate chemolithoautotrophic bacterium of the phylum Aquificae and is capable of fixing carbon dioxide through the reductive tricarboxylic acid (TCA) cycle. The recent discovery of two novel-type phosphoserine phosphatases (PSPs) in H. thermophilus suggests the presence of a phosphorylated serine biosynthesis pathway; however, the physiological role of these novel-type metal-independent PSPs (iPSPs) in H. thermophilus has not been confirmed. In the present study, a mutant strain with a deletion of pspA , the catalytic subunit of iPSPs, was constructed and characterized. The generated mutant was a serine auxotroph, suggesting that the novel-type PSPs and phosphorylated serine synthesis pathway are essential for serine anabolism in H. thermophilus. As an autotrophic medium supplemented with glycine did not support the growth of the mutant, the reversible enzyme serine hydroxymethyltransferase does not appear to synthesize serine from glycine and may therefore generate glycine and 5,10-CH 2 -tetrahydrofolate (5,10-CH 2 -THF) from serine. This speculation is supported by the lack of glycine cleavage activity, which is needed to generate 5,10-CH 2 -THF, in H. thermophilus Determining the mechanism of 5,10-CH 2 -THF synthesis is important for understanding the fundamental anabolic pathways of organisms, because 5,10-CH 2 -THF is a major one-carbon donor that is used for the synthesis of various essential compounds, including nucleic and amino acids. The findings from the present experiments using a pspA deletion mutant have confirmed the physiological role of iPSPs as serine producers and show that serine is a major donor of one-carbon units in H. thermophilus IMPORTANCE Serine biosynthesis and catabolism pathways are intimately related to the metabolism of 5,10-CH 2 -THF, a one-carbon donor that is utilized for the biosynthesis of various essential compounds. For this reason, determining the mechanism of serine synthesis is important for understanding the fundamental anabolic pathways of microorganisms. In the present study, we experimentally confirmed that a novel phosphoserine phosphatase in the obligate chemolithoautotrophic bacterium Hydrogenobacter thermophilus is essential for serine biosynthesis. This finding indicates that serine is synthesized from an intermediate of gluconeogenesis in H. thermophilus In addition, because glycine cleavage system activity and genes encoding an enzyme capable of producing 5,10-CH 2 -THF were not detected, serine appears to be the major one-carbon donor to tetrahydrofolate (THF) in H. thermophilus . Copyright © 2017 American Society for Microbiology.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

PubMed

Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

Stress inducible proteinase inhibitor diversity in Capsicum annuum

PubMed Central

2012-01-01

Background Wound-inducible Pin-II Proteinase inhibitors (PIs) are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L.) proteinase inhibitor (CanPIs) genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS) of Helicoverpa armigera or water. Results The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs). Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and −10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and −43. Conclusions Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack. PMID:23153298

The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

PubMed

Scott, Glynis; Leopardi, Sonya; Parker, Lorelle; Babiarz, Laura; Seiberg, Miri; Han, Rujiing

2003-09-01

Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

PubMed

Sin, Suk-Fong; Chye, Mee-Len

2004-10-01

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

Targeting Inhibition of Fibroblast Activation Protein-α and Prolyl Oligopeptidase Activities on Cells Common to Metastatic Tumor Microenvironments1

PubMed Central

Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N; Downs, Tamyra D; McKee, Patrick A

2013-01-01

Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth. PMID:23555181

Lactobacillus helveticus as a tool to change proteolysis and functionality in Swiss-type cheeses.

PubMed

Sadat-Mekmene, L; Richoux, R; Aubert-Frogerais, L; Madec, M-N; Corre, C; Piot, M; Jardin, J; le Feunteun, S; Lortal, S; Gagnaire, V

2013-03-01

Lactobacillus helveticus exhibits a great biodiversity in terms of protease gene content, with 1 to 4 cell envelope proteinases. Among them, proteinases PrtH and PrtH2 were shown to have different cleavage specificity on pure α(s1)-casein. The aim of this work was to investigate the proteolytic activity of 2L. helveticus strains in cheese matrix: ITGLH77 (PrtH2 only) and ITGLH1 (at least 2 proteinases, PrtH and PrtH2). Cell viability, proteolysis, autolysis, and stretchability of experimental Emmental cheeses were measured during ripening. The peptides identified by mass spectrometry showed very different profiles in the 2 cheeses. Regardless of the casein origin, the number of different peptides containing more than 20 amino acids was greater in cheeses manufactured with strain ITGLH77. This accumulation of large peptides, including those from α(s1)- and α(s2)-caseins, was in agreement with the lower overall extent of proteolysis obtained in ITGLH77 cheeses, which can be attributed to the presence of one cell envelope proteinase of the lactobacilli strains or lesser release of intracellular peptidases into the cheese aqueous phase. In parallel, stretchability was measured throughout ripening time. Emmental strands observed by confocal laser scanning microscopy showed microstructure similar to that of mozzarella strands. Stretchability was correlated with a specific type of peptide (hydrophobic), as shown by principal component analysis, and with a lower degree of proteolysis. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Protocol to obtain targeted transcript sequence data from snake venom samples collected in the Colombian field.

PubMed

Fonseca, Alejandra; Renjifo-Ibáñez, Camila; Renjifo, Juan Manuel; Cabrera, Rodrigo

2018-03-21

Snake venoms are a mixture of different molecules that can be used in the design of drugs for various diseases. The study of these venoms has relied on strategies that use complete venom extracted from animals in captivity or from venom glands that require the sacrifice of the animals. Colombia, a country with political and geographical conflicts has difficult access to certain regions. A strategy that can prevent the sacrifice of animals and could allow the study of samples collected in the field is necessary. We report the use of lyophilized venom from Crotalus durissus cumanensis as a model to test, for the first time, a protocol for the amplification of complete toxins from Colombian venom samples collected in the field. In this protocol, primers were designed from conserved region from Crotalus sp. mRNA and EST regions to maximize the likelihood of coding sequence amplification. We obtained the sequences of Metalloproteinases II, Disintegrins, Disintegrin-Like, Phospholipases A 2, C-type Lectins and Serine proteinases from Crotalus durissus cumanensis and compared them to different Crotalus sp sequences available on databases obtaining concordance between the toxins amplified and those reported. Our strategy allows the use of lyophilized venom to obtain complete toxin sequences from samples collected in the field and the study of poorly characterized venoms in challenging environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Granzyme B mediates both direct and indirect cleavage of extracellular matrix in skin after chronic low-dose ultraviolet light irradiation.

PubMed

Parkinson, Leigh G; Toro, Ana; Zhao, Hongyan; Brown, Keddie; Tebbutt, Scott J; Granville, David J

2015-02-01

Extracellular matrix (ECM) degradation is a hallmark of many chronic inflammatory diseases that can lead to a loss of function, aging, and disease progression. Ultraviolet light (UV) irradiation from the sun is widely considered as the major cause of visible human skin aging, causing increased inflammation and enhanced ECM degradation. Granzyme B (GzmB), a serine protease that is expressed by a variety of cells, accumulates in the extracellular milieu during chronic inflammation and cleaves a number of ECM proteins. We hypothesized that GzmB contributes to ECM degradation in the skin after UV irradiation through both direct cleavage of ECM proteins and indirectly through the induction of other proteinases. Wild-type and GzmB-knockout mice were repeatedly exposed to minimal erythemal doses of solar-simulated UV irradiation for 20 weeks. GzmB expression was significantly increased in wild-type treated skin compared to nonirradiated controls, colocalizing to keratinocytes and to an increased mast cell population. GzmB deficiency significantly protected against the formation of wrinkles and the loss of dermal collagen density, which was related to the cleavage of decorin, an abundant proteoglycan involved in collagen fibrillogenesis and integrity. GzmB also cleaved fibronectin, and GzmB-mediated fibronectin fragments increased the expression of collagen-degrading matrix metalloproteinase-1 (MMP-1) in fibroblasts. Collectively, these findings indicate a significant role for GzmB in ECM degradation that may have implications in many age-related chronic inflammatory diseases. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The role played by serine proteases in the development and worsening of vascular complications in type 1 diabetes mellitus.

PubMed

Finotti, Paola

2006-08-01

Much attention has been given to the role played by serine proteases in the development and worsening of vascular complications in Type 1 diabetes mellitus. A generalized increase in proteolytic activity, either due to a true increase in concentration of specific proteases or defects of their protease inhibitors, represents an early marker of diabetes. However, the precise molecular mechanism whereby an unopposed proteolytic activity leads to overt vascular alterations has not fully been elucidated as yet. The picture is further complicated by the fact that, although sharing the same function, serine proteases constitute a structurally heterogeneous class of molecules. Besides classical proteases, for most part belonging to coagulative and fibrinolytic systems, other unrelated molecules exhibit serine-like protease activity and are capable of triggering both inflammatory and immune reactions. The specific role of these non classical serine proteases in the complex pathogenesis of diabetes and its vascular complications is attracting a new investigative interest, as these molecules may represent additional therapeutic targets. This review will focus on most recent acquisitions on this issue relevant to Type 1 diabetes.

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

PubMed

Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

1996-01-16

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

PubMed Central

Nakayama, K; Yoshimura, F; Kadowaki, T; Yamamoto, K

1996-01-01

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins. PMID:8631669

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

PubMed Central

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha

2015-01-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. PMID:25870228

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

PubMed

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

2015-07-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases.

PubMed

Cox, S W; Eley, B M; Kiili, M; Asikainen, A; Tervahartiala, T; Sorsa, T

2006-01-01

Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.

Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

PubMed Central

Gay, Carolina; Sanz, Libia; Calvete, Juan J.; Pla, Davinia

2015-01-01

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families, being dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom. PMID:26712790

The amino acid sequences of carboxypeptidases I and II from Aspergillus niger and their stability in the presence of divalent cations.

PubMed

Svendsen, I; Dal Degan, F

1998-09-08

The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants

PubMed Central

Rodrigues, Silas P.; Ventura, José A.; Aguilar, Clemente; Nakayasu, Ernesto S.; Choi, HyungWon; Sobreira, Tiago J. P.; Nohara, Lilian L.; Wermelinger, Luciana S.; Almeida, Igor C.; Zingali, Russolina B.; Fernandes, Patricia M. B.

2012-01-01

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers’ regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

Phylogenomic evolutionary surveys of subtilase superfamily genes in fungi.

PubMed

Li, Juan; Gu, Fei; Wu, Runian; Yang, JinKui; Zhang, Ke-Qin

2017-03-30

Subtilases belong to a superfamily of serine proteases which are ubiquitous in fungi and are suspected to have developed distinct functional properties to help fungi adapt to different ecological niches. In this study, we conducted a large-scale phylogenomic survey of subtilase protease genes in 83 whole genome sequenced fungal species in order to identify the evolutionary patterns and subsequent functional divergences of different subtilase families among the main lineages of the fungal kingdom. Our comparative genomic analyses of the subtilase superfamily indicated that extensive gene duplications, losses and functional diversifications have occurred in fungi, and that the four families of subtilase enzymes in fungi, including proteinase K-like, Pyrolisin, kexin and S53, have distinct evolutionary histories which may have facilitated the adaptation of fungi to a broad array of life strategies. Our study provides new insights into the evolution of the subtilase superfamily in fungi and expands our understanding of the evolution of fungi with different lifestyles.

Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kutsyi, M.P.; Gaziev, A.I.

An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after {gamma} irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiothreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis thatmore » several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats. 43 refs., 7 figs.« less

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

PubMed

De Oliveira Neto, Osmundo Brilhante; Batista, João Aguiar Nogueira; Rigden, Daniel John; Franco, Octávio Luiz; Fragoso, Rodrigo Rocha; Monteiro, Ana Carolina Santos; Monnerat, Rose Gomes; Grossi-De-Sa, Maria Fátima

2004-06-01

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.

Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases.

PubMed

Alderete, J F; Newton, E; Dennis, C; Neale, K A

1991-08-01

A recent report demonstrated the immunogenic character of the cysteine proteinases of Trichomonas vaginalis. It was of interest, therefore, to examine for the presence of serum anti-proteinase antibody among patients with trichomoniasis. An immunoprecipitation assay was used involving protein A-bearing Staphylococcus aureus first coated with the IgG fraction of goat anti-human Ig and then mixed with individual sera of patients to bind human antibody. These antibody-coated bacteria were then added to detergent extracts of T vaginalis. Bound immune complexes on S aureus were washed and solubilised for electrophoretic analysis on acrylamide copolymerised with gelatin for detection of proteinase activity. Sera from patients (50/50), but none from sera of normal, uninfected women, possessed IgG to numerous trichomonad cysteine proteinases. The presence of this serum anti-proteinase antibody disappeared after drug treatment and cure of patients of the T vaginalis infection. The commonality of the anti-proteinase antibody in the sera of patients with trichomoniasis provided evidence for the expression of the same repertoire of parasite proteinases during infection. These observations have important implications for the in vivo relevance of the proteinases and indicate that strategies to use a specific serum antibody response for diagnosis of this infection may be possible.

Inhibition of trypanosomal cysteine proteinases by their propeptides.

PubMed

Lalmanach, G; Lecaille, F; Chagas, J R; Authié, E; Scharfstein, J; Juliano, M A; Gauthier, F

1998-09-25

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15-mer peptides covering the whole prosequence of congopain, the major cysteine proteinase of Trypanosoma congolense. Three consecutive peptides with a common 5-mer sequence YHNGA were competitive inhibitors of congopain. A shorter synthetic peptide consisting of this 5-mer sequence flanked by two Ala residues (AYHNGAA) also inhibited purified congopain. No residue critical for inhibition was identified in this sequence, but a significant improvement in Ki value was obtained upon N-terminal elongation. Procongopain-derived peptides did not inhibit lysosomal cathepsins B and L but did inhibit native cruzipain (from Dm28c clone epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The positioning of the YHNGA inhibitory sequence within the prosegment of trypanosomal proteinases is similar to that covering the active site in the prosegment of cysteine proteinases, the three-dimensional structure of which has been resolved. This strongly suggests that trypanosomal proteinases, despite their long C-terminal extension, have a prosegment that folds similarly to that in related mammal and plant cysteine proteinases, resulting in reverse binding within the active site. Such reverse binding could also occur for short procongopain-derived inhibitory peptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, homologous peptides in related cysteine proteinases did not inhibit trypanosomal proteinases and were rapidly cleaved by these enzymes.

Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

PubMed Central

Peng, Chih-Wen; Peremyslov, Valera V.; Mushegian, Arcady R.; Dawson, William O.; Dolja, Valerian V.

2001-01-01

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event. PMID:11711606

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

PubMed

Peng, C W; Peremyslov, V V; Mushegian, A R; Dawson, W O; Dolja, V V

2001-12-01

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

The role of proteinase 3 (PR3) and the protease-activated receptor-2 (PAR-2) pathway in dendritic cell (DC) maturation of human-DC-like monocytes and murine DC.

PubMed

Jiang, Bo; Grage-Griebenow, Evelin; Csernok, Elena; Butherus, Kristine; Ehlers, Stefan; Gross, Wolfgang L; Holle, Julia U

2010-01-01

The aim of the study was to assess PAR-2 expression on dendritic cell (DC) subsets and other immune cells of Wegener's granulomatosis (WG) patients and healthy controls (HC) and to investigate whether Proteinase 3 (PR3, a serine protease which can activate PAR2) induces maturation of human DC-like monocytes and murine Flt-3 ligand- and GM-CSF-generated DC. Human peripheral blood cells including DC subsets and Flt-3l- and GM-CSF-generated mouse DC were analysed for expression of PAR-2 and DC maturation markers by flow cytometry before and after stimulation with PR3, trypsin, PAR-2 agonist or LPS for 24 h. There was no difference of PAR-2 expression on PMNs, monocytes, lymphocytes and DC between all WG samples and HC. However, in inactive WG, expression of PAR-2 was downregulated on the cell surface of PMNs, monocytes, lymphocytes, and CD11c+DC compared to active WG and HC. PR3 and PAR2-agonists did not induce upregulation of PAR-2 or maturation markers of human DC-like monocytes in WG and HC. Likewise, murine PR3 did not induce upregulation of PAR-2 or maturation markers in murine DC. PAR-2 expression is downregulated on human peripheral blood cells including CD11c+ DC in inactive WG compared to active WG and HC, possibly reflecting a non-activated status of these cells in inactive disease. PR3 and PAR-2- agonists did not induce maturation of human ex vivo DC-like monocytes in WG and HC and of murine DC, suggesting this pathway is not singularly involved in the maturation of these cell subsets.

Involvement of proteinase activated receptor-2 in the vascular response to sphingosine 1-phosphate.

PubMed

Roviezzo, Fiorentina; De Angelis, Antonella; De Gruttola, Luana; Bertolino, Antonio; Sullo, Nikol; Brancaleone, Vincenzo; Bucci, Mariarosaria; De Palma, Raffaele; Urbanek, Konrad; D'Agostino, Bruno; Ianaro, Angela; Sorrentino, Raffaella; Cirino, Giuseppe

2014-04-01

S1P (sphingosine 1-phosphate) represents one of the key latest additions to the list of vasoactive substances that modulate vascular tone. PAR-2 (proteinase activated receptor-2) has been shown to be involved in cardiovascular function. In the present study, we investigated the involvement of PAR-2 in S1P-induced effect on vascular tone. The present study has been performed by using isolated mouse aortas. Both S1P and PAR-2 agonists induced endothelium-dependent vasorelaxation. L-NAME (N(G)-nitro-L-arginine methyl ester) and wortmannin abrogated the S1P-induced vasorelaxatioin, while significantly inhibiting the PAR-2-mediated effect. Either ENMD1068, a PAR-2 antagonist, or gabexate, a serine protease inhibitor, significantly inhibited S1P-induced vasorelaxation. Aortic tissues harvested from mice overexpressing PAR-2 displayed a significant increase in vascular response to S1P as opposed to PAR-2-null mice. Immunoprecipitation and immunofluorescence studies demonstrated that S1P(1) interacted with PAR-2 and co-localized with PAR-2 on the vascular endothelial surface. Furthermore, S1P administration to vascular tissues triggered PAR-2 mobilization from the plasma membrane to the perinuclear area; S1P-induced translocation of PAR-2 was abrogated when aortic rings were pre-treated with ENMD1068 or when caveolae dysfunction occurred. Similarly, experiments performed in cultured endothelial cells (human umbilical vein endothelial cells) showed a co-localization of S1P(1) and PAR2, as well as the ability of S1P to induce PAR-2 trafficking. Our results suggest that S1P induces endothelium-dependent vasorelaxation mainly through S1P(1) and involves PAR-2 transactivation.

High Fidelity Processing and Activation of the Human α-Defensin HNP1 Precursor by Neutrophil Elastase and Proteinase 3

PubMed Central

Tongaonkar, Prasad; Golji, Amir E.; Tran, Patti; Ouellette, André J.; Selsted, Michael E.

2012-01-01

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1–4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (“folded proHNP1”) and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro. PMID:22448222

High fidelity processing and activation of the human α-defensin HNP1 precursor by neutrophil elastase and proteinase 3.

PubMed

Tongaonkar, Prasad; Golji, Amir E; Tran, Patti; Ouellette, André J; Selsted, Michael E

2012-01-01

The azurophilic granules of human neutrophils contain four α-defensins called human neutrophil peptides (HNPs 1-4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages ("folded proHNP1") and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.

Proteomic endorsed transcriptomic profiles of venom glands from Tityus obscurus and T. serrulatus scorpions

PubMed Central

Nishiyama, Milton Yutaka; dos Santos, Maria Beatriz Viana; Santos-da-Silva, Andria de Paula; Chalkidis, Hipócrates de Menezes; Souza-Imberg, Andreia; Candido, Denise Maria; Yamanouye, Norma; Dorce, Valquíria Abrão Coronado; Junqueira-de-Azevedo, Inácio de Loiola Meirelles

2018-01-01

Background Except for the northern region, where the Amazonian black scorpion, T. obscurus, represents the predominant and most medically relevant scorpion species, Tityus serrulatus, the Brazilian yellow scorpion, is widely distributed throughout Brazil, causing most envenoming and fatalities due to scorpion sting. In order to evaluate and compare the diversity of venom components of Tityus obscurus and T. serrulatus, we performed a transcriptomic investigation of the telsons (venom glands) corroborated by a shotgun proteomic analysis of the venom from the two species. Results The putative venom components represented 11.4% and 16.7% of the total gene expression for T. obscurus and T. serrulatus, respectively. Transcriptome and proteome data revealed high abundance of metalloproteinases sequences followed by sodium and potassium channel toxins, making the toxin core of the venom. The phylogenetic analysis of metalloproteinases from T. obscurus and T. serrulatus suggested an intraspecific gene expansion, as we previously observed for T. bahiensis, indicating that this enzyme may be under evolutionary pressure for diversification. We also identified several putative venom components such as anionic peptides, antimicrobial peptides, bradykinin-potentiating peptide, cysteine rich protein, serine proteinases, cathepsins, angiotensin-converting enzyme, endothelin-converting enzyme and chymotrypsin like protein, proteinases inhibitors, phospholipases and hyaluronidases. Conclusion The present work shows that the venom composition of these two allopatric species of Tityus are considerably similar in terms of the major classes of proteins produced and secreted, although their individual toxin sequences are considerably divergent. These differences at amino acid level may reflect in different epitopes for the same protein classes in each species, explaining the basis for the poor recognition of T. obscurus venom by the antiserum raised against other species. PMID:29561852

A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

PubMed Central

Hussain, Haitham; Branny, Pavel; Allan, Elaine

2006-01-01

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis. PMID:16452447

Airway Fibrinogenolysis and the Initiation of Allergic Inflammation

PubMed Central

Millien, Valentine Ongeri; Lu, Wen; Mak, Garbo; Yuan, Xiaoyi; Knight, J. Morgan; Porter, Paul; Kheradmand, Farrah

2014-01-01

The past 15 years of allergic disease research have produced extraordinary improvements in our understanding of the pathogenesis of airway allergic diseases such as asthma. Whereas it was previously viewed as largely an immunoglobulin E-mediated process, the gradual recognition that T cells, especially Type 2 T helper (Th2) cells and Th17 cells, play a major role in asthma and related afflictions has inspired clinical trials targeting cytokine-based inflammatory pathways that show great promise. What has yet to be clarified about the pathogenesis of allergic inflammatory disorders, however, are the fundamental initiating factors, both exogenous and endogenous, that drive and sustain B- and T-cell responses that underlie the expression of chronic disease. Here we review how proteinases derived from diverse sources drive allergic responses. A central discovery supporting the proteinase hypothesis of allergic disease pathophysiology is the role played by airway fibrinogen, which in part appears to serve as a sensor of unregulated proteinase activity and which, when cleaved, both participates in a novel allergic signaling pathway through Toll-like receptor 4 and forms fibrin clots that contribute to airway obstruction. Unresolved at present is the ultimate source of airway allergenic proteinases. From among many potential candidates, perhaps the most intriguing is the possibility such enzymes derive from airway fungi. Together, these new findings expand both our knowledge of allergic disease pathophysiology and options for therapeutic intervention. PMID:25525732

Assessing the level of matrix metal proteinases 1,8,9, their tissue inhibitor, type I, in cases of odontogenic phlegmons.

PubMed

Markelova, E V; Romanchuk, A L; Prosekova, E V; Krasnikov, V E; Beniova, S N

2017-01-01

The article considers the measured values of the level of MMP-1, MMP-8 and MMP-9, and of their tissue inhibitor Type I (TIMP-1) in the blood serum and mixed saliva samples of 78 patients (31 women - 36.2 %, 47 men - 63.8 %) suffering from odontogenic phlegmons in the oral and maxillofacial region. The study indicators were assessed through the enzyme-linked immunosorbent assay using diagnostic sets RandD Diagnostics Inc. (USA). An uncontrolled hyperactivation of metal proteinases as one of the components of the systemic inflammatory response in case of phlegmon-related complications in the oral and maxillofacial region, as well as development of the sepsis syndrome were studied and it was determined that it results in distortion of the processes of reparative hystogeny and compel us to employ new approaches to the treatment of this type of patients (Tab. 2, Fig. 1, Ref. 13).

Molecular investigation on the interaction of spermine with proteinase K by multispectroscopic techniques and molecular simulation studies.

PubMed

Hosseini-Koupaei, Mansoore; Shareghi, Behzad; Saboury, Ali Akbar; Davar, Fateme

2017-01-01

The alteration in structure, function and stability of proteinase K in the presence of spermine was investigated using spectroscopic methods and simulation techniques. The stability and enzyme activity of proteinase K-spermine complex were significantly enhanced as compared to that of the pure enzyme. The increase in the value of V max and the catalytic efficiency of Proteinase K in presence of spermine confirmed that the polyamine could bring the enzyme hyperactivation. UV-vis spectroscopy, intrinsic fluorescence and circular dichroism methods demonstrated that the binding of spermine changed the microenvironment and structure of proteinase K. The fluorescence studies, showing that spermine quenched the intensity of proteinase K with static mechanism. Thermodynamic parameters analysis suggested that hydrogen bond and van der Waals forces play a key role in complex stability which is in agreement with modeling studies. The CD spectra represented the secondary structure alteration of proteinase K with an increase in α-helicity and a decrease in β-sheet of proteinase K upon spermine conjugation. The molecular simulation results proposed that spermine could interact with proteinase K spontaneously at single binding site, which is in agreement with spectroscopic results. This agreement between experimental and theoretical results may be a worth method for protein-ligand complex studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomic and biological characterization of the venom of the redtail coral snake, Micrurus mipartitus (Elapidae), from Colombia and Costa Rica.

PubMed

Rey-Suárez, Paola; Núñez, Vitelbina; Gutiérrez, José María; Lomonte, Bruno

2011-12-21

Venoms of the redtail coral snake Micrurus mipartitus from Colombia and Costa Rica were analyzed by "venomics", a proteomic strategy to determine their composition. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI or ESI tandem mass spectrometry, and assignment to known protein families by similarity. These analyses were complemented with a characterization of venom activities in vitro and in vivo. Proteins belonging to seven families were found in Colombian M. mipartitus venom, including abundant three-finger toxins (3FTx; ~60% of total proteins) and phospholipases A(2) (PLA(2); ~30%), with the remaining ~10% distributed among l-amino acid oxidase, P-III metalloproteinase, Kunitz-type inhibitor, serine proteinase, and C-type lectin-like families. The venoms of two M. mipartitus specimens from Costa Rica, also referred to as M. multifasciatus in some taxonomic classifications, were also analyzed. Both samples were highly similar to each other, and partially resembled the chromatographic and identity profiles of M. mipartitus from Colombia, although presenting a markedly higher proportion of 3FTxs (~83.0%) in relation to PLA(2)s (~8.2%), and a small amount of acetylcholinesterase, not detected in the venom from Colombia. An equine antivenom against the Central American coral snake, M. nigrocinctus, did not recognize venom components of M. mipartitus from Colombia or Costa Rica by enzyme-immunoassay. Four major components of Colombian M. mipartitus venom were isolated and partially characterized. Venomics of Micrurus species may provide a valuable platform for the rational design of immunizing cocktails to obtain polyspecific antivenoms for this highly diverse group of American elapids. Copyright © 2011 Elsevier B.V. All rights reserved.

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

PubMed Central

Sukuru, Sai Chetan K; Nigsch, Florian; Quancard, Jean; Renatus, Martin; Chopra, Rajiv; Brooijmans, Natasja; Mikhailov, Dmitri; Deng, Zhan; Cornett, Allen; Jenkins, Jeremy L; Hommel, Ulrich; Davies, John W; Glick, Meir

2010-01-01

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors. PMID:20799349

Serine deprivation enhances antineoplastic activity of biguanides.

PubMed

Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

2014-12-15

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. ©2014 American Association for Cancer Research.

The skin barrier function gene SPINK5 is associated with challenge-proven IgE-mediated food allergy in infants.

PubMed

Ashley, S E; Tan, H-T T; Vuillermin, P; Dharmage, S C; Tang, M L K; Koplin, J; Gurrin, L C; Lowe, A; Lodge, C; Ponsonby, A-L; Molloy, J; Martin, P; Matheson, M C; Saffery, R; Allen, K J; Ellis, J A; Martino, D

2017-09-01

A defective skin barrier is hypothesized to be an important route of sensitization to dietary antigens and may lead to food allergy in some children. Missense mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) skin barrier gene have previously been associated with allergic conditions. To determine whether genetic variants in and around SPINK5 are associated with IgE-mediated food allergy. We genotyped 71 "tag" single nucleotide polymorphisms (tag-SNPs) within a region spanning ~263 kb including SPINK5 (~61 kb) in n=722 (n=367 food-allergic, n=199 food-sensitized-tolerant and n=156 non-food-allergic controls) 12-month-old infants (discovery sample) phenotyped for food allergy with the gold standard oral food challenge. Transepidermal water loss (TEWL) measures were collected at 12 months from a subset (n=150) of these individuals. SNPs were tested for association with food allergy using the Cochran-Mantel-Haenszel test adjusting for ancestry strata. Association analyses were replicated in an independent sample group derived from four paediatric cohorts, total n=533 (n=203 food-allergic, n=330 non-food-allergic), mean age 2.5 years, with food allergy defined by either clinical history of reactivity, 95% positive predictive value (PPV) or challenge, corrected for ancestry by principal components. SPINK5 variant rs9325071 (A⟶G) was associated with challenge-proven food allergy in the discovery sample (P=.001, OR=2.95, CI=1.49-5.83). This association was further supported by replication (P=.007, OR=1.58, CI=1.13-2.20) and by meta-analysis (P=.0004, OR=1.65). Variant rs9325071 is associated with decreased SPINK5 gene expression in the skin in publicly available genotype-tissue expression data, and we generated preliminary evidence for association of this SNP with elevated TEWL also. We report, for the first time, association between SPINK5 variant rs9325071 and challenge-proven IgE-mediated food allergy. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Structural features of diverse Pin-II proteinase inhibitor genes from Capsicum annuum.

PubMed

Mahajan, Neha S; Dewangan, Veena; Lomate, Purushottam R; Joshi, Rakesh S; Mishra, Manasi; Gupta, Vidya S; Giri, Ashok P

2015-02-01

The proteinase inhibitor (PI) genes from Capsicum annuum were characterized with respect to their UTR, introns and promoter elements. The occurrence of PIs with circularly permuted domain organization was evident. Several potato inhibitor II (Pin-II) type proteinase inhibitor (PI) genes have been analyzed from Capsicum annuum (L.) with respect to their differential expression during plant defense response. However, complete gene characterization of any of these C. annuum PIs (CanPIs) has not been carried out so far. Complete gene architectures of a previously identified CanPI-7 (Beads-on-string, Type A) and a member of newly isolated Bracelet type B, CanPI-69 are reported in this study. The 5' UTR (untranslated region), 3'UTR, and intronic sequences of both the CanPI genes were obtained. The genomic sequence of CanPI-7 exhibited, exon 1 (49 base pair, bp) and exon 2 (740 bp) interrupted by a 294-bp long type I intron. We noted the occurrence of three multi-domain PIs (CanPI-69, 70, 71) with circularly permuted domain organization. CanPI-69 was found to possess exon 1 (49 bp), exon 2 (551 bp) and a 584-bp long type I intron. The upstream sequence analysis of CanPI-7 and CanPI-69 predicted various transcription factor-binding sites including TATA and CAAT boxes, hormone-responsive elements (ABRELATERD1, DOFCOREZM, ERELEE4), and a defense-responsive element (WRKY71OS). Binding of transcription factors such as zinc finger motif MADS-box and MYB to the promoter regions was confirmed using electrophoretic mobility shift assay followed by mass spectrometric identification. The 3' UTR analysis for 25 CanPI genes revealed unique/distinct 3' UTR sequence for each gene. Structures of three domain CanPIs of type A and B were predicted and further analyzed for their attributes. This investigation of CanPI gene architecture will enable the better understanding of the genetic elements present in CanPIs.

Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dougherty, W.

1995-10-01

The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function inmore » a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.« less

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

d-serine levels in Alzheimer's disease: implications for novel biomarker development

PubMed Central

Madeira, C; Lourenco, M V; Vargas-Lopes, C; Suemoto, C K; Brandão, C O; Reis, T; Leite, R E P; Laks, J; Jacob-Filho, W; Pasqualucci, C A; Grinberg, L T; Ferreira, S T; Panizzutti, R

2015-01-01

Alzheimer's disease (AD) is a severe neurodegenerative disorder still in search of effective methods of diagnosis. Altered levels of the NMDA receptor co-agonist, d-serine, have been associated with neurological disorders, including schizophrenia and epilepsy. However, whether d-serine levels are deregulated in AD remains elusive. Here, we first measured D-serine levels in post-mortem hippocampal and cortical samples from nondemented subjects (n=8) and AD patients (n=14). We next determined d-serine levels in experimental models of AD, including wild-type rats and mice that received intracerebroventricular injections of amyloid-β oligomers, and APP/PS1 transgenic mice. Finally, we assessed d-serine levels in the cerebrospinal fluid (CSF) of 21 patients with a diagnosis of probable AD, as compared with patients with normal pressure hydrocephalus (n=9), major depression (n=9) and healthy controls (n=10), and results were contrasted with CSF amyloid-β/tau AD biomarkers. d-serine levels were higher in the hippocampus and parietal cortex of AD patients than in control subjects. Levels of both d-serine and serine racemase, the enzyme responsible for d-serine production, were elevated in experimental models of AD. Significantly, d-serine levels were higher in the CSF of probable AD patients than in non-cognitively impaired subject groups. Combining d-serine levels to the amyloid/tau index remarkably increased the sensitivity and specificity of diagnosis of probable AD in our cohort. Our results show that increased brain and CSF d-serine levels are associated with AD. CSF d-serine levels discriminated between nondemented and AD patients in our cohort and might constitute a novel candidate biomarker for early AD diagnosis. PMID:25942042

Mutation of the Kunitz-type proteinase inhibitor domain in the amyloid β-protein precursor abolishes its anti-thrombotic properties in vivo.

PubMed

Xu, Feng; Davis, Judianne; Hoos, Michael; Van Nostrand, William E

2017-07-01

Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid β-protein precursor (AβPP) inhibit cerebral thrombosis. KPI domain-lacking forms of AβPP are abundant in brain. Regions of AβPP other than the KPI domain may also be involved with regulating cerebral thrombosis. To determine the contribution of the KPI domain to the overall function of AβPP in regulating cerebral thrombosis we generated a reactive center mutant that was devoid of anti-thrombotic activity and studied its anti-thrombotic function in vitro and in vivo. To determine the extent of KPI function of AβPP in regulating cerebral thrombosis we generated a recombinant reactive center KPI R13I mutant devoid of anti-thrombotic activity. The anti-proteolytic and anti-coagulant properties of wild-type and R13I mutant KPI were investigated in vitro. Cerebral thrombosis of wild-type, AβPP knock out and AβPP/KPI R13I mutant mice was evaluated in experimental models of carotid artery thrombosis and intracerebral hemorrhage. Recombinant mutant KPI R13I domain was ineffective in the inhibition of pro-thrombotic proteinases and did not inhibit the clotting of plasma in vitro. AβPP/KPI R13I mutant mice were similarly deficient as AβPP knock out mice in regulating cerebral thrombosis in experimental models of carotid artery thrombosis and intracerebral hemorrhage. We demonstrate that the anti-thrombotic function of AβPP primarily resides in the KPI activity of the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteinase pattern in Trametes versicolor in response to carbon and nitrogen starvation.

PubMed

Staszczak, M; Nowak, G

1984-01-01

In stationary cultures of Trametes versicolor seven proteinase bands were revealed by electrophoresis in mycelium and five in the medium. Under conditions of nitrogen starvation the number of bands in mycelium was unchanged whereas one extracellular proteinase was missing. In the case of carbon starvation one new intracellular proteinase activity appeared and one extracellular activity disappeared. Moreover, in all starved cultures distinct differences in the intensity of particular bands were observed.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

PubMed

Almeida, Carla Malaquias; Pereira, Cláudia; da Costa, Diana Soares; Pereira, Susana; Pissarra, José; Simões, Isaura; Faro, Carlos

2012-07-01

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia.

PubMed

Søreide, Kjetil

2008-08-01

Of all the body systems, the gastrointestinal (GI) tract is the most exposed to proteinases. Proteolytic activity must thus be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological GI conditions. The protease-activated receptor-2 (PAR-2) is expressed in numerous cell types within the GI tract, suggesting both multiple functions and numerous modes of receptor activation. Although best known as a pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers. Of interest, trypsin and PAR-2 act together in an autocrine loop that promotes proliferation, invasion and metastasis in neoplasia through various mechanisms. Trypsin and PAR-2 seem to act both directly and indirectly through activation of other proteinase cascades, including metalloproteinases. PAR-2 activation can participate in inflammatory reactions, be protective to mucosal surfaces, send or inhibit nociceptive messages, modify gut motility or secretory functions, and stimulate cell proliferation and motility. Several studies point to a role for the PARs in disease processes of the GI tract and pancreas ranging from inflammatory bowel disease, symptoms associated with irritable bowel syndrome, pain in pancreatitis, development of colon and other GI cancers, and even infectious colitis. Proteinases should not only be considered from the traditional view as digestive or degradative enzymes in the gut, but additionally as signalling molecules that actively participate in the spectrum of physiology and diseased states of the GI tract.

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

PubMed Central

Prabhu, V; Chatson, K B; Abrams, G D; King, J

1996-01-01

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment. PMID:8819325

Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

PubMed Central

2012-01-01

Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes. PMID:22380654

Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

PubMed Central

Richards, Ryan; Dempski, Robert E.

2012-01-01

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2. PMID:23185520

Transmembrane Segment Five Serines of the D4 Dopamine Receptor Uniquely Influence the Interactions of Dopamine, Norepinephrine, and Ro10-4548

PubMed Central

Cummings, David F.; Ericksen, Spencer S.; Goetz, Angela

2010-01-01

Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with α1- and α2-adrenergic, β2-adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant and had reduced potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. In contrast, Ro10-4548 [RAC-2′-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide], a catechol-like antagonist of the wild-type receptor unexpectedly functions as an agonist of the S5.43A mutant. Other noncatechol ligands had similar properties for mutant and wild-type receptors. This is the first example of a dopamine receptor point mutation selectively changing the receptor's interaction with a specific antagonist to that of an agonist, and together with other data, provides evidence, supported by molecular modeling, that catecholamine-type agonism is induced by different ligand-specific configurations of intermolecular H-bonds with the TM5 conserved serines. PMID:20215412

[Activity of tissue cathepsin-L-like proteinases of women with womb body oncopathology].

PubMed

Vovchuk, I L; Chernadchuk, S S

2004-01-01

Activity and optimal pH of cathepsin-L-like proteinases was studied in benign and malignant tumours of the womb body. In the benign tumors activity of cathepsin-L-like proteinases changes depending on the expansion and depth of extension benign tumour and is defined by proliferative potential of tumour cells of myometrium and endometrium. Activity of cathepsin-L-like proteinases in malignant epithelial tumour of endometrium--adenocarcinoma is inversely proportional to the level of differentiation of the tumour cells.

[Lactic acid bacteria proteinase and quality of fermented dairy products--A review].

PubMed

Zhang, Shuang; Zhang, Lanwei; Han, Xue

2015-12-04

Lactic acid bacteria (LAB) could synthesize cell envelope proteinase with weak activity, which primarily degrades casein. In addition to its crucial role in the rapid growth of LAB in milk, LAB proteinases are also of industrial importance due to their contribution to the formation of texture and flavor of many fermented dairy products. The proteolytic system, properties of proteinase, the degradation product of casein and its effect on the quality of fermented dairy products were reviewed in this manuscript.

Alpha-1 antitrypsin reduces ovariectomy-induced bone loss in mice

USDA-ARS?s Scientific Manuscript database

Alpha-1antitrypsin (AAT) is a multifunctional protein with proteinase inhibitor and anti-inflammatory activities. Recent studies showed that AAT has therapeutic effect for diseases associated with inflammation, such as type 1 diabetes and arthritis. Proinflammatory cytokines are primary mediators of...

Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents.

PubMed

Ozkan, Semiha; Kaynak, Fatma; Kalkanci, Ayse; Abbasoglu, Ufuk; Kustimur, Semra

2005-05-01

Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinase activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

PAR-2 receptor-induced effects on human eccrine sweat gland cells.

PubMed

L Bovell, Douglas; Kofler, Barbara; Lang, Roland

2009-01-01

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Protease-activated receptor-2 (PAR(2)) in human periodontitis.

PubMed

Holzhausen, M; Cortelli, J R; da Silva, V Araújo; Franco, G C Nobre; Cortelli, S Cavalca; Vergnolle, N

2010-09-01

No evidence for the role of protease-activated receptor-2 (PAR(2)) in human periodontal disease has been demonstrated so far. Thus, we sought to investigate the expression of PAR(2) mRNA in chronic periodontitis, and to examine whether its expression is related to the presence of PAR(2) potential activators. Microbiological and gingival crevicular fluid samples were collected from individuals with chronic periodontitis and control individuals, and the presence of neutrophil serine proteinase 3 (P3) and Porphyromonas gingivalis was evaluated. PAR(2) mRNA expression was higher (p < 0.001) in those with chronic periodontitis compared with control individuals, and it was statistically decreased (p = 0.0006) after periodontal treatment. Furthermore, those with chronic periodontitis presented higher (p < 0.05) levels of IL-1alpha, IL-6, IL-8, and TNF-alpha, total proteolytic activity, P. gingivalis prevalence, and P3mRNA expression compared with control individuals. We conclude that PAR(2) mRNA expression and its potential activators are elevated in human chronic periodontitis, therefore suggesting that PAR(2) may play a role in periodontal inflammation.

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.

PubMed

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

2012-06-18

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis and functional evaluation of a peptide derivative of 1-beta-D-arabinofuranosylcytosine.

PubMed

Balajthy, Z; Aradi, J; Kiss, I T; Elödi, P

1992-09-04

We have synthesized a peptidyl prodrug derivative of 1-beta-D-arabinofuranosylcytosine (1) designed to be a selective substrate of plasmin. D-Val-Leu-Lys-ara-C (2) was obtained by coupling the protected peptide Cbz-D-Val-Leu-(N6-Cbz)Lys-OH and ara-C (1) by a water-soluble carbodiimide (EDCI), followed by the removal of the Cbz groups by using catalytic hydrogenolysis over Pd/C. The kinetic constant of hydrolysis of 2 in the presence of plasmin demonstrated effective release of 1. The amino group of 1, which is sensitive to the removal by cytidine deaminase, is protected in 2 by the formation of the amide bond resulting in a prolonged half-life of 2 in biological milieu. The antiproliferative efficiency of 2 against L1210 leukemic cells was significantly higher than that of 1. The activity of 2 was abolished in the presence of serine proteinase inhibitor, (4-amidinopheny)methanesulfonyl fluoride. These data indicate that 2 is a prodrug form of 1 in systems generating plasmin.

Neutral endopeptidase inhibitors potentiate substance P-induced contraction in gut smooth muscle.

PubMed

Djokic, T D; Sekizawa, K; Borson, D B; Nadel, J A

1989-01-01

To determine the role of endogenous neutral endopeptidase (NEP), also called enkephalinase (EC 3.4.24.11), in regulating tachykinin-induced contraction of gut smooth muscle, we studied the effects of NEP inhibitors on the contractile responses to substance P (SP) in isolated longitudinal strips of ileum or duodenum in rats and ferrets. Leucine-thiorphan and phosphoramidon shifted the concentration-response curves of SP to lower concentrations in all tissues studied, but the sensitivity to SP was greater and the effect of leucine-thiorphan was less in the ferret, a finding that correlated with the observation that the ferret ileum contained substantially less NEP activity than rat ileum. Captopril, bestatin, MGTA, leupeptin, and physostigmine did not alter contractile responses to SP, suggesting that kininase II, aminopeptidases, carboxypeptidase N, serine proteinases, and acetylcholinesterase do not modulate the SP-induced effects. These studies suggest that, in the ileum and duodenum, NEP modulates the actions of SP and, furthermore, that the sensitivity of tissues may be determined, at least in part, by the amount of enzymatically active NEP present.

Responses of prophenoloxidase system and related defence parameters of Litopenaeus vannamei to low salinity

NASA Astrophysics Data System (ADS)

Pan, Luqing; Xie, Peng; Hu, Fawen

2010-09-01

In this study, we investigated the effects of low salinity (26 and 21) on the prophenoloxidase (proPO) system and related defence parameters in the shrimp Litopenaeus vannamei. The results showed that low salinity induced a significant increase of dopamine (DA) concentration in haemolymph at 6 h of the experiment; on the other hand, total haemocyte count (THC), differential haemocyte count (DHC) and PO activity decreased over time to the lowest level at 24 h and remained low thereafter. Serine Protease (SP) and Proteinase Inhibitor (PI) activity in the two lower salinity treatments decreased to the lowest level at 12 and 24 h, respectively, and both recovered to the control level at 72 h. In contrast, α2- macroglobulin (α2M) activity in the two lower salinity treatments peaked at 24 h and then decreased to the control level at 72 h. Therefore, it may be concluded that stress-induced DA plays an important temporary role in neurotransmission and causes immune response in L. vannamei in adapting to salinity changes.

Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus).

PubMed

Pedra, Joao H F; Brandt, Amanda; Li, Hong-Mei; Westerman, Rick; Romero-Severson, Jeanne; Pollack, Richard J; Murdock, Larry L; Pittendrigh, Barry R

2003-11-01

Genomics information relating to human body lice is surprisingly scarce, and this has constrained studies of their physiology, immunology and vector biology. To identify novel body louse genes, we used engorged adult lice to generate a cDNA library. Initially, 1152 clones were screened for inserts, edited for removal of vector sequences and base pairs of poor quality, and viewed for splicing variations, gene families and polymorphism. Computational methods identified 506 inferred open reading frames including the first predicted louse defensin. The inferred defensin aligns well with other insect defensins and has highly conserved cysteine residues, as are known for other defensin sequences. Two cysteine and five serine proteinases were categorized according to their inferred catalytic sites. We also discovered seven putative ubiquitin-pathway genes and four iron metabolizing deduced enzymes. Finally, glutathione-S-transferases and cytochrome P450 genes were among the detoxification enzymes found. Results from this first systematic effort to discover human body louse genes should promote further studies in Phthiraptera and lice.

Inhibitory and antimicrobial activities of OGTI and HV-BBI peptides, fragments and analogs derived from amphibian skin.

PubMed

Dębowski, Dawid; Łukajtis, Rafał; Łęgowska, Anna; Karna, Natalia; Pikuła, Michał; Wysocka, Magdalena; Maliszewska, Irena; Sieńczyk, Marcin; Lesner, Adam; Rolka, Krzysztof

2012-06-01

A series of linear and cyclic fragments and analogs of two peptides (OGTI and HV-BBI) isolated from skin secretions of frogs were synthesized by the solid-phase method. Their inhibitory activity against several serine proteinases: bovine β-trypsin, bovine α-chymotypsin, human leukocyte elastase and cathepsin G from human neutrophils, was investigated together with evaluation of their antimicrobial activities against Gram-negative bacteria (Escherichia coli) and Gram-positive species isolated from patients (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus sp., Streptococcus sp.). The cytotoxicity of the selected peptides toward an immortal human skin fibroblast cell line was also determined. Three peptides: HV-BBI, its truncated fragment HV-BBI(3-18) and its analog [Phe(8)]HV-BBI can be considered as bifunctional compounds with inhibitory as well as antibacterial properties. OGTI, although it did not display trypsin inhibitory activity as previously reported in the literature, exerted antimicrobial activity toward S. epidermidis. In addition, under our experimental conditions, this peptide did not show cytotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

PubMed

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

PubMed Central

Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

2013-01-01

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown. PMID:23150644

Adult Schistosoma mansoni express cathepsin L proteinase activity.

PubMed

Smith, A M; Dalton, J P; Clough, K A; Kilbane, C L; Harrop, S A; Hole, N; Brindley, P J

1994-09-01

This report presents the deduced amino acid sequence of a novel cathepsin L proteinase from Schistosoma mansoni, and describes cathepsin L-like activity in extracts of adult schistosomes. Using consensus primers specific for cysteine proteinases, gene fragments were amplified from adult S. mansoni cDNA by PCR and cloned. One of these fragments showed marked identity to Sm31, the cathepsin B cysteine proteinase of adult S. mansoni, whereas another differed from Sm31 and was employed as a probe to isolate two cDNAs from an adult S. mansoni gene library. Together these cDNAs encoded a novel preprocathepsin L of 319 amino acids; this zymogen is predicted to be processed in vivo into a mature, active cathepsin L proteinase of 215 amino acids. Closest homologies were with cathepsins L from rat, mouse, and chicken (46-47% identity). Southern hybridization analysis suggested that only one or a few copies of the gene was present per genome, demonstrated that its locus was distinct from that of Sm31, and that a homologous sequence was present in Schistosoma japonicum. Because these results indicated that schistosomes expressed a cathepsin L proteinase, extracts of adult S. mansoni were examined for acidic, cysteine proteinase activity. Based on rates of cleavage of peptidyl substrates employed to discriminate between classes of cysteine proteinases, namely cathepsin L (Z-phe-arg-AMC), cathepsin B (Z-arg-arg-AMC) and cathepsin H (Bz-arg-AMC), the extracts were found to contain vigorous cathepsin L-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Small Molecule Inhibitors of microRNA Involved in Chemoresistance and Cancer Stem Cells for Ovarian Cancer Intervention

DTIC Science & Technology

2013-09-01

reversion-inducing-cysteine- rich protein with kazal motifs (RECK) in oral cancer . J Biol Chem. 287:29261-72. 2012 3. Lin FC, Liu YP, Lai CH, Shan YS...Annual Meeting, 2012. 8. Jung HM, Patel RS, Cohen DM, Jakymiw A, Kong WM, Cheng JQ, Chan EKL. Subclassification of oral cancers using a miRNA-based

Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review.

PubMed

Oliva, Maria Luiza Vilela; Ferreira, Rodrigo da Silva; Ferreira, Joana Gasperazzo; de Paula, Cláudia Alessandra Andrade; Salas, Carlos E; Sampaio, Misako Uemura

2011-08-01

Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, G.N.M.; Knowles, N.R.; Houtz, R.L.

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as in increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-inducedmore » increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed daughter tubers that contained 3-fold more protein than mother tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Their results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; the authors also identify a role for PMC in regulating protein turnover in potato tubers.« less

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei.

PubMed

Hernandez-Cortes, Patricia; Rivera-Pérez, Crisalejandra; García-Carreño, Fernando; Martínez-Alarcón, Diana

2017-02-01

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

THE CRYSTALLIZATION AND SEROLOGICAL DIFFERENTIATION OF A STREPTOCOCCAL PROTEINASE AND ITS PRECURSOR

PubMed Central

Elliott, S. D.

1950-01-01

Grown in dialysate broth at a pH between 5.5 and 6.5, some strains of group A streptococci elaborate the precursor of a proteolytic enzyme. Within this range of hydrogen concentration the precursor is also produced when the streptococci are suspended in a peptone dialysate containing glucose and incubated at 37°C. The precursor does not appear to be produced at a neutral or alkaline reaction. Methods are described whereby the precursor and proteinase have been isolated in crystalline form. The precursor crystallizes from half-saturated ammonium sulfate at pH 8.0 and a temperature of 22°C. or higher; the proteinase crystallizes from 0.15 saturated ammonium sulfate at pH 8.0 but does so most readily at refrigerator temperature. The degree of purification achieved by these procedures is discussed. The activity of purified preparations of the precursor and of proteinase has been tested against α-benzoyl-l-arginineamide and, with this as a substrate, the conversion of precursor to proteinase by autocatalysis or by trypsin has been confirmed. Immunological experiments are described, the results of which provide evidence of the distinct antigenic specificity of the precursor and proteinase; the conversion of precursor to proteinase has been followed by means of serological tests. PMID:15436931

Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

PubMed

Wei, Xiumei; Xu, Jie; Yang, Jianmin; Liu, Xiangquan; Zhang, Ranran; Wang, Weijun; Yang, Jialong

2015-01-01

Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of two serpins of Clonorchis sinensis by bioinformatics, expression, and localization in metacercaria

PubMed Central

Yang, Yabo; Hu, Dong; Wang, Lexun; Liang, Chi; Hu, Xuchu; Xu, Jin; Huang, Yan; Yu, Xinbing

2014-01-01

Clonorchiasis, which has been an important public health problem in China, is caused by ingestion of raw or undercooked fish contaminated by live metacercaria. Therefore, preventing fish from infecting is of great significance for controlling the disease. SERPINs (serine protease inhibitors) are well known as negative regulators of hemostasis, thrombolysis, and innate immune responses. In the present study, two full-length sequences encoding SERPIN were identified from metacercaria cDNA library of Clonorchis sinensis (C. sinensis) and were denominated as CsSERPIN and CsSERPIN3, respectively. Bioinformatics analysis showed that the two sequences shares 35.9% identity to each other. Both of the sequences have SERPIN domain and the greatest difference between the two domains is the reactive centre loop. Transmembrane region was found in CsSERPIN3 while not in CsSERPIN. The expression of the two CsSERPINs was significantly higher at the life stage of metacercaria than that of adult. The transcription levels of CsSERPIN and CsSERPIN3 at metacercaria stage were 3.249- and 11.314-fold of that at adult stage, respectively. Furthermore, the expression of CsSERPIN was 4.32-fold of that of CsSERPIN3 at metacercaria stage. Immunobiochemistry revealed that CsERPIN was dispersed at subtegument and oral sucker of metacercaria, while CsSERPIN3 localized intensely in the tegument of metacercaria of C. sinensis inside of the cyst wall. All these indicated that the CsSERPINs play important roles at metacercaria stage of the parasite. CsSERPIN may take part in regulation of endogenous serine proteinase and CsSERPIN3 may be involved in immune evasion and be a potential candidate for vaccine and drug target for clonorchiasis. PMID:24831344

Comparison of two serpins of Clonorchis sinensis by bioinformatics, expression, and localization in metacercaria.

PubMed

Yang, Yabo; Hu, Dong; Wang, Lexun; Liang, Chi; Hu, Xuchu; Xu, Jin; Huang, Yan; Yu, Xinbing

2014-06-01

Clonorchiasis, which has been an important public health problem in China, is caused by ingestion of raw or undercooked fish contaminated by live metacercaria. Therefore, preventing fish from infecting is of great significance for controlling the disease. SERPINs (serine protease inhibitors) are well known as negative regulators of hemostasis, thrombolysis, and innate immune responses. In the present study, two full-length sequences encoding SERPIN were identified from metacercaria cDNA library of Clonorchis sinensis (C. sinensis) and were denominated as CsSERPIN and CsSERPIN3, respectively. Bioinformatics analysis showed that the two sequences shares 35.9% identity to each other. Both of the sequences have SERPIN domain and the greatest difference between the two domains is the reactive centre loop. Transmembrane region was found in CsSERPIN3 while not in CsSERPIN. The expression of the two CsSERPINs was significantly higher at the life stage of metacercaria than that of adult. The transcription levels of CsSERPIN and CsSERPIN3 at metacercaria stage were 3.249- and 11.314-fold of that at adult stage, respectively. Furthermore, the expression of CsSERPIN was 4.32-fold of that of CsSERPIN3 at metacercaria stage. Immunobiochemistry revealed that CsERPIN was dispersed at subtegument and oral sucker of metacercaria, while CsSERPIN3 localized intensely in the tegument of metacercaria of C. sinensis inside of the cyst wall. All these indicated that the CsSERPINs play important roles at metacercaria stage of the parasite. CsSERPIN may take part in regulation of endogenous serine proteinase and CsSERPIN3 may be involved in immune evasion and be a potential candidate for vaccine and drug target for clonorchiasis.

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism.

PubMed

Ge, Shuqing; Li, Tao; Yao, Qijian; Yan, Hongling; Huiyun, Zhang; Zheng, Yanshan; Zhang, Bin; He, Shaoheng

2016-12-01

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2 %. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310 % increase in TNF-α release from monocytes at 16 h, respectively. PAR-1 agonist peptide, SFLLR-NH 2 , and PAR-2 agonist peptide tc-LIGRLO-NH 2 provoked up to 210 and 240 % increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH 2 -induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH 2 , an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH 2 -induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroach major allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

PubMed

Faller, Nicolas; Gautschi, Ivan; Schild, Laurent

2014-01-01

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor

NASA Astrophysics Data System (ADS)

Goto, Masaru

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

Transcripts of sulphur metabolic genes are co-ordinately regulated in developing seeds of common bean lacking phaseolin and major lectins

PubMed Central

Marsolais, Frédéric

2012-01-01

The lack of phaseolin and phytohaemagglutinin in common bean (dry bean, Phaseolus vulgaris) is associated with an increase in total cysteine and methionine concentrations by 70% and 10%, respectively, mainly at the expense of an abundant non-protein amino acid, S-methyl-cysteine. Transcripts were profiled between two genetically related lines differing for this trait at four stages of seed development using a high density microarray designed for common bean. Transcripts of multiple sulphur-rich proteins were elevated, several previously identified by proteomics, including legumin, basic 7S globulin, albumin-2, defensin, albumin-1, the Bowman–Birk type proteinase inhibitor, the double-headed trypsin inhibitor, and the Kunitz trypsin inhibitor. A co-ordinated regulation of transcripts coding for sulphate transporters, sulphate assimilatory enzymes, serine acetyltransferases, cystathionine β-lyase, homocysteine S-methyltransferase and methionine gamma-lyase was associated with changes in cysteine and methionine concentrations. Differential gene expression of sulphur-rich proteins preceded that of sulphur metabolic enzymes, suggesting a regulation by demand from the protein sink. Up-regulation of SERAT1;1 and -1;2 expression revealed an activation of cytosolic O-acetylserine biosynthesis. Down-regulation of SERAT2;1 suggested that cysteine and S-methyl-cysteine biosynthesis may be spatially separated in different subcellular compartments. Analysis of free amino acid profiles indicated that enhanced cysteine biosynthesis was correlated with a depletion of O-acetylserine. These results contribute to our understanding of the regulation of sulphur metabolism in developing seed in response to a change in the composition of endogenous proteins. PMID:23066144

Transcripts of sulphur metabolic genes are co-ordinately regulated in developing seeds of common bean lacking phaseolin and major lectins.

PubMed

Liao, Dengqun; Pajak, Agnieszka; Karcz, Steven R; Chapman, B Patrick; Sharpe, Andrew G; Austin, Ryan S; Datla, Raju; Dhaubhadel, Sangeeta; Marsolais, Frédéric

2012-10-01

The lack of phaseolin and phytohaemagglutinin in common bean (dry bean, Phaseolus vulgaris) is associated with an increase in total cysteine and methionine concentrations by 70% and 10%, respectively, mainly at the expense of an abundant non-protein amino acid, S-methyl-cysteine. Transcripts were profiled between two genetically related lines differing for this trait at four stages of seed development using a high density microarray designed for common bean. Transcripts of multiple sulphur-rich proteins were elevated, several previously identified by proteomics, including legumin, basic 7S globulin, albumin-2, defensin, albumin-1, the Bowman-Birk type proteinase inhibitor, the double-headed trypsin inhibitor, and the Kunitz trypsin inhibitor. A co-ordinated regulation of transcripts coding for sulphate transporters, sulphate assimilatory enzymes, serine acetyltransferases, cystathionine β-lyase, homocysteine S-methyltransferase and methionine gamma-lyase was associated with changes in cysteine and methionine concentrations. Differential gene expression of sulphur-rich proteins preceded that of sulphur metabolic enzymes, suggesting a regulation by demand from the protein sink. Up-regulation of SERAT1;1 and -1;2 expression revealed an activation of cytosolic O-acetylserine biosynthesis. Down-regulation of SERAT2;1 suggested that cysteine and S-methyl-cysteine biosynthesis may be spatially separated in different subcellular compartments. Analysis of free amino acid profiles indicated that enhanced cysteine biosynthesis was correlated with a depletion of O-acetylserine. These results contribute to our understanding of the regulation of sulphur metabolism in developing seed in response to a change in the composition of endogenous proteins.

Immunization with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I (CPB) and Type II (CPA), partially protects against leishmaniasis.

PubMed

Zadeh-Vakili, Azita; Taheri, Tahere; Taslimi, Yasaman; Doustdari, Fatemeh; Salmanian, Ali-Hatef; Rafati, Sima

2004-05-07

Cysteine proteinases (CPs) are enzymes that belong to the papain superfamily, which are found in a number of organisms from prokaryotes to mammals. On the parasitic protozoan Leishmania, extensive studies have shown that CPs are involved in parasite survival, replication and the onset of disease, and have, therefore, been considered as attractive drugs and/or vaccine targets for the control of leishmaniasis. We have previously shown that cysteine proteinases, Type I (CPB) and Type II (CPA), in Leishmania major (L. major), delivered as recombinant proteins or in plasmid DNA, induce partial protection against infection with the parasite in BALB/c mice. We had shown that the level of protection was greater if a cocktail of cpa and cpb containing DNA constructs was used. Therefore, to reduce the costs associated with the production of these vaccine candidates, a construct was developed, whereby the cpa and cpb genes were fused together to give rise to a single hybrid protein. The genes were fused in tandem where the C-terminal extension (CTE), encoding region of CPB, was located at the 3' of the fused genes, and ultimately expressed in the bacterial expression construct pET-23a. The expression of the CPA/B hybrid protein (60 kDa) was verified using rabbit anti-CPA and anti-CPB antibodies by SDS-PAGE and immunoblotting. The protective potential of the CPA/B hybrid protein against the infection with Leishmania was then assessed in BALB/c mice. The animals were vaccinated with CPA/B, challenged with live L. major promastigotes, and the degree of protection was examined by measuring footpad lesion sizes. It was found that there was a delay in the expansion of lesions size compared to control groups. Furthermore, an immunological analysis of antibody isotypes, before and after infection, showed high levels of IgG2a compared to IgG1 (more than five-fold) in the CPA/B hybrid protein vaccinated group. In addition, a predominant Th1 immune response characterized by in vitro IFN-gamma production was observed, along with little, if any, IL-5 production. This finding indicates that the hybrid CPA/B is able to elicit a protective immune response against L. major in the mice model. In addition, 54% of individuals tested, who had recovered from cutaneous leishmaniasis, produced more than 50 pg/ml IFN-gamma, in response to the CPA/B hybrid protein in an in vitro assay, demonstrating the importance of cysteine proteinases as targets of immune response in humans.

Enterovirus Control of Translation and RNA Granule Stress Responses.

PubMed

Lloyd, Richard E

2016-03-30

Enteroviruses such as poliovirus (PV) and coxsackievirus B3 (CVB3) have evolved several parallel strategies to regulate cellular gene expression and stress responses to ensure efficient expression of the viral genome. Enteroviruses utilize their encoded proteinases to take over the cellular translation apparatus and direct ribosomes to viral mRNAs. In addition, viral proteinases are used to control and repress the two main types of cytoplasmic RNA granules, stress granules (SGs) and processing bodies (P-bodies, PBs), which are stress-responsive dynamic structures involved in repression of gene expression. This review discusses these processes and the current understanding of the underlying mechanisms with respect to enterovirus infections. In addition, the review discusses accumulating data suggesting linkage exists between RNA granule formation and innate immune sensing and activation.

A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence

PubMed Central

Connolly, James P. R.; Gabrielsen, Mads; Goldstone, Robert J.; Grinter, Rhys; Wang, Dai; Cogdell, Richard J.; Walker, Daniel; Smith, David G. E.; Roe, Andrew J.

2016-01-01

The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to “sense” levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host. PMID:26727373

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

PubMed Central

Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

1987-01-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

Phospholipase and proteinase activities of Candida spp. isolates from vulvovaginitis in Iran.

PubMed

Shirkhani, S; Sepahvand, A; Mirzaee, M; Anbari, K

2016-09-01

This study aims to characterize phospholipase and proteinase activities of Candida isolates from 82 vulvovaginal candidiasis (VVC) and to study the relationship of these activities with vulvovaginitis. Totally 82 Candida isolates from vagina samples of VVC patients were randomly collected over the period between September and December 2014 from hospitalized patients at the general hospitals of Lorestan province, Iran. Isolates were previously identified by conventional mycological methods. The phospholipase and proteinase activities were evaluated by Egg yolk agar, Tween 80 opacity medium and agar plate methods. The most common Candida species was identified Candida albicans (n=34, 41.5%), followed by Candida famata (n=13, 15.8%), Candida tropicalis (n=11, 13.4%), and Candida parapsilosis (n=9, 11%). The most phospholipase activity was observed in Candida colliculosa (40%), followed by C. famata (38.5%), and Candida krusei (33.3%). The findings revealed that the correlation between phospholipase production by Candida spp. and the presence of VVC was not found to be statistically significant (P=0.91). All Candida spp. exhibited considerable proteinase activity; so that 100% of C. colliculosa, C. parapsilosis, Candida kefyr, and Candida intermedia isolates produced high proteinase activity with Pz 4+ scores. There was a significant correlation between proteinase production by Candida spp. and the presence of VVC (P=0.009). The obtained findings revealed that Candida spp. isolates may produce both virulence factors, phospholipase and proteinase. Although the phospholipase production was only observed in <40% of the isolates; however there was a significant association between proteinase production by Candida spp. and VVC. Copyright © 2016. Published by Elsevier Masson SAS.

PAR(2) expression in peripheral blood monocytes of patients with rheumatoid arthritis.

PubMed

Crilly, A; Burns, E; Nickdel, M B; Lockhart, J C; Perry, M E; Ferrell, P W; Baxter, D; Dale, J; Dunning, L; Wilson, H; Nijjar, J S; Gracie, J A; Ferrell, W R; McInnes, I B

2012-06-01

Proteinase-activated receptor 2 (PAR(2)) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functional significance of PAR(2) expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR(2) on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Patients with RA had elevated but variable surface expression of PAR(2) on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86-4.10%) vs 0.06% (0.03-0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR(2) expression in patients with RA compared with controls (3.05% (0.36-11.82%) vs 0.08% (0.02-0.28%), p<0.0001). For both subsets, PAR(2) expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR(2) expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR(2) expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR(2) expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR(2) agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). These findings are consistent with a pathogenic role for PAR(2) in RA.

Metabolism of native and naturally occurring multiple modified low density lipoprotein in smooth muscle cells of human aortic intima.

PubMed

Tertov, V V; Orekhov, A N

1997-01-01

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.

PAR2 expression in peripheral blood monocytes of patients with rheumatoid arthritis

PubMed Central

Crilly, A; Burns, E; Nickdel, M B; Lockhart, J C; Perry, M E; Ferrell, P W; Baxter, D; Dale, J; Dunning, L; Wilson, H; Nijjar, J S; Gracie, J A; Ferrell, W R; McInnes, I B

2012-01-01

Objectives Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functional significance of PAR2 expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Methods Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR2 on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Results Patients with RA had elevated but variable surface expression of PAR2 on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86–4.10%) vs 0.06% (0.03–0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR2 expression in patients with RA compared with controls (3.05% (0.36–11.82%) vs 0.08% (0.02–0.28%), p<0.0001). For both subsets, PAR2 expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR2 expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR2 expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR2 expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR2 agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). Conclusions These findings are consistent with a pathogenic role for PAR2 in RA. PMID:22294633

Occurrence and Distribution of Proteinase of Streptococcus faecalis var. liquefaciens1

PubMed Central

Shugart, Lee R.; Beck, Raymond W.

1966-01-01

Shugart, Lee R. (University of Tennessee, Knoxville), and Raymond W. Beck. Occurrence and distribution of proteinase of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 92:338–341. 1966.—The proteolytic enzyme produced by Streptococcus faecalis var. liquefaciens (ATCC 13398) was shown to be an exoenzyme. The production of the proteinase was followed in growing cultures, and its distribution was compared with that of the intracellular enzymes reduced nicotinamide adenine dinucleotide (NADH2) peroxidase and lactate dehydrogenase. The proteinase appeared in the culture medium prior to the stationary phase of growth, whereas the other enzymes could be found only in whole cells. Fractionation of whole cells by sonic treatment and by treatment with lysozyme showed the proteinase to be associated primarily with the cell wall and cell membrane, and NADH2 peroxidase to be associated only with the cytoplasmic fractions. PMID:16562116

Pathogenesis-Related Proteins of Tomato 1

PubMed Central

Vera, Pablo; Conejero, Vicente

1988-01-01

An endoproteinase induced by citrus exocortis viroid has been purified from tomato (Lycopersicon esculentum Mill, cv “Rutgers”) leaves. The proteinase corresponds to one of the major pathogenesis-related proteins of tomato plants and was designated proteinase P-69 as it has a molecular weight of 69,000 to 70,000. The proteinase was purified in four steps: (NH4)2SO4 fractionation, chromatography on Bio-Gel P-60, DEAE-Sepharose chromatography, and casein-Sepharose affinity chromatography. The proteinase had a pH optimum of 8.5 to 9.0 when assayed with either fluorescein thiocarbamoyl derivative (FTC)-casein or FTC-ribulose 1,5-bisphosphate carboxylase/oxygenase as substrates. The proteinase activity was inhibited by pCMB and strongly activated by calcium and magnesium ions as well as by DTT. When analyzed by electrofocusing, the activity showed a pI around 9.0. Images Fig. 4 Fig. 8 PMID:16666127

A lysosomal pepstatin-insensitive proteinase as a novel biomarker for breast carcinoma.

PubMed

Junaid, M A; Clark, G M; Pullarkat, R K

2000-01-01

Lysosomal proteinases play an important role in the turnover of intracellular proteins, and acidic proteinases such as cathepsin D are known to be increased in breast carcinoma. In the present study the activity of a newly discovered acidic lysosomal pepstatin-insensitive proteinase (CLN2p) was measured in breast tissues by the most sensitive and highly specific assay that we had developed for the diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) (2). Samples from eight normal subjects undergoing reductive mammoplasty and 200 patients with primary breast carcinoma were analyzed. The results suggest a two- to seventeen-fold higher CLN2p activity in tumors, which was significantly and positively correlated with already known breast cancer biomarkers such as levels of cathepsin D, estrogen receptor and progesterone receptor. These results suggest a diagnostic and prognostic potential for this novel acid proteinase in breast cancer.

Lowering plasma 1-deoxysphingolipids improves neuropathy in diabetic rats.

PubMed

Othman, Alaa; Bianchi, Roberto; Alecu, Irina; Wei, Yu; Porretta-Serapiglia, Carla; Lombardi, Raffaella; Chiorazzi, Alessia; Meregalli, Cristina; Oggioni, Norberto; Cavaletti, Guido; Lauria, Giuseppe; von Eckardstein, Arnold; Hornemann, Thorsten

2015-03-01

1-Deoxysphingolipids (1-deoxySLs) are atypical neurotoxic sphingolipids that are formed by the serine-palmitoyltransferase (SPT). Pathologically elevated 1-deoxySL concentrations cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonal neuropathy associated with several missense mutations in SPT. Oral L-serine supplementation suppressed the formation of 1-deoxySLs in patients with HSAN1 and preserved nerve function in an HSAN1 mouse model. Because 1-deoxySLs also are elevated in patients with type 2 diabetes mellitus, L-serine supplementation could also be a therapeutic option for diabetic neuropathy (DN). This was tested in diabetic STZ rats in a preventive and therapeutic treatment scheme. Diabetic rats showed significantly increased plasma 1-deoxySL concentrations, and L-serine supplementation lowered 1-deoxySL concentrations in both treatment schemes (P < 0.0001). L-serine had no significant effect on hyperglycemia, body weight, or food intake. Mechanical sensitivity was significantly improved in the preventive (P < 0.01) and therapeutic schemes (P < 0.001). Nerve conduction velocity (NCV) significantly improved in only the preventive group (P < 0.05). Overall NCV showed a highly significant (P = 5.2E-12) inverse correlation with plasma 1-deoxySL concentrations. In summary, our data support the hypothesis that 1-deoxySLs are involved in the pathology of DN and that an oral L-serine supplementation could be a novel therapeutic option for treating DN. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

PubMed

Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

1998-08-25

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

Mosaic serine proteases in the mammalian central nervous system.

PubMed

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

PubMed

Waern, Ida; Karlsson, Iulia; Thorpe, Michael; Schlenner, Susan M; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Åbrink, Magnus; Hellman, Lars; Pejler, Gunnar; Wernersson, Sara

2012-12-01

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

PubMed Central

Loo, Shining; Kam, Antony; Xiao, Tianshu; Nguyen, Giang K. T.; Liu, Chuan Fa; Tam, James P.

2016-01-01

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases. PMID:27991569

Metabolism of hydroxypyruvate in a mutant of barley lacking NADH-dependent hydroxypyruvate reductase, an important photorespiratory enzyme activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, A.J.S.; Blackwell, R.D.; Lea, P.J.

1989-09-01

A mutant of barley (Hordeum vulgare L.), LaPr 88/29, deficient in NADH-dependent hydroxypyruvate reductase (HPR) activity has been isolated. The activities of both NADH (5%) and NADPH-dependent (19%) HPR were severely reduced in this mutant compared to the wild type. Although lacking an enzyme in the main carbon pathway of photorespiration, this mutant was capable of CO{sub 2} fixation rates equivalent to 75% of that of the wild type, in normal atmospheres and 50% O{sub 2}. There also appeared to be little disruption to the photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{supmore » 14}C)serine feeding were similar in both mutant and wild-type leaves. When leaves of LaPr 88/29 were fed either ({sup 14}C)serine or {sup 14}CO{sub 2}, the accumulation of radioactivity was in serine and not in hydroxypyruvate, although the mutant was still able to metabolize over 25% of the supplied ({sup 14}C)serine into sucrose. After 3 hours in air the soluble amino acid pool was almost totally dominated by serine and glycine. LaPr 88/29 has also been used to show that NADH-glyoxylate reductase and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-dependent HPR activity is due to the NADH-dependent enzyme. We also suggest that the alternative NADPH activity can metabolize a proportion, but not all, of the hydroxypyruvate produced during photorespiration and may thus form a useful backup to the NADH-dependent enzyme under conditions of maximal photorespiration.« less

Crystal structure of the NADP+ and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

PubMed

Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

2018-05-01

A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP + /sulfate ion at 1.18 Å and the structure in complex with NADP + /L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP + and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.

Recognition of microbial molecular patterns and stimulation of prophenoloxidase activation by a β-1,3-glucanase-related protein in Manduca sexta larval plasma

PubMed Central

Wang, Yang; Sumathipala, Niranji; Rayaprolu, Subrahmanyam; Jiang, Haobo

2011-01-01

Detection of pathogenic invaders is the essential first step of a successful defense response in multicellular organisms. In this study, we have identified a new member of the β-1,3-glucanase-related protein superfamily from the tobacco hornworm Manduca sexta. This protein, designated microbe binding protein (MBP), is 61% identical in sequence to Bombyx mori Gram-negative bacteria binding protein, but only 34-36% identical to M. sexta β-1,3-glucan recognition protein-1 and 2. Its mRNA levels were strongly up-regulated in hemocytes and fat body of immune challenged larvae, along with an increase in concentration of the plasma protein. We expressed M. sexta MBP in a baculovirus-insect cell system. The purified protein associated with intact bacteria and fungi. It specifically bound to lipoteichoic acid, lipopolysaccharide, diaminopimelic acid-type peptidoglycans (DAP-PGs) from Escherichia coli and Bacillus subtilis, but less so to laminarin or Lys-type PG from Staphylococcus aureus. The complex binding pattern was influenced by other plasma factors and additional microbial surface molecules. After different amounts of MBP had been incubated with larval plasma on ice, a concentration-dependent increase in phenoloxidase (PO) activity occurred in the absence of any microbial elicitor. The activity increase was also observed in the mixture of plasma and a bacterial or fungal cell wall component. The prophenoloxidase (proPO) activation became more prominent when DAP-PGs, Micrococcus luteus Lys-PG, or lipoteichoic acid was included in the mixture of MBP and plasma. Statistic analysis suggested that a synergistic enhancement of proPO activation was caused by an interaction between MBP and these elicitors, but not S. aureus Lys-PG, lipopolysaccharide, curdlan, or laminarin. These data indicate that M. sexta MBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and serine proteinases, triggers the proPO activation system. PMID:21296155

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

PubMed Central

Blainey, Paul C.; Graziano, Vito; Pérez-Berná, Ana J.; McGrath, William J.; Flint, S. Jane; San Martín, Carmen; Xie, X. Sunney; Mangel, Walter F.

2013-01-01

Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 106 bp2/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA. PMID:23043138

Exploring Hydrophobic Binding Surfaces Using Comfa and Flexible Hydrophobic Ligands

NASA Astrophysics Data System (ADS)

Thakkar, Shraddha; Sanchez, Rosa. I.; Bhuveneswaran, Chidambaram; Compadre, Cesar M.

2011-06-01

Cysteine proteinases are a very important group of enzymes involved in a variety of physiological and pathological processes including cancer metastasis and rheumatoid arthritis. In this investigation we used 3D-Quantitative Structure Activity Relationships (3D-QSAR) techniques to model the binding of a variety of substrates to two cysteine proteinases, papain, and cathepsin B. The analysis was performed using Comparative Molecular Field Analysis (CoMFA). The molecules were constructed using standard bond angles and lengths, minimized and aligned. Charges were calculated using the PM3 method in MOPAC. The CoMFA models derived for the binding of the studied substrates to the two proteinases were compared with the expected results from the experimental X-ray crystal structures of the same proteinases. The results showed the value of CoMFA modeling of flexible hydrophobic ligands to analyze ligand binding to protein receptors, and could also serve as the basis to design specific inhibitors of cysteine proteinases with potential therapeutic value.

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

PubMed Central

Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

1987-01-01

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer. PMID:16347298

Proteolytic Cascade for the Activation of the Insect Toll Pathway Induced by the Fungal Cell Wall Component

PubMed Central

Roh, Kyung-Baeg; Kim, Chan-Hee; Lee, Hanna; Kwon, Hyun-Mi; Park, Ji-Won; Ryu, Ji-Hwan; Kurokawa, Kenji; Ha, Nam-Chul; Lee, Won-Jae; Lemaitre, Bruno; Söderhäll, Kenneth; Lee, Bok-Luel

2009-01-01

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-κB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Recently, we identified three serine proteases mediating Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative-binding protein 3 (GNBP3), a receptor for a major cell wall component β-1,3-glucan of fungi, and their order of activation have not been characterized yet. Here, we identified three serine proteases that are required for Toll activation by β-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, we show that injection of β-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal β-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation. PMID:19473968

The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

NASA Technical Reports Server (NTRS)

Rojas, Ana; Doolittle, Russell F.

2003-01-01

Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

Functional characterization of single-domain cystatin-like cysteine proteinase inhibitors expressed by the trematode Fasciola hepatica.

PubMed

Cancela, Martín; Corvo, Ileana; DA Silva, Edileuza; Teichmann, Aline; Roche, Leda; Díaz, Alvaro; Tort, José Fransisco; Ferreira, Henrique B; Zaha, Arnaldo

2017-11-01

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

PubMed

Wei, Wei; Gao, Yanhui; Wang, Cheng; Zhao, Lijun; Sun, Dianjun

2013-06-01

Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis. Copyright © 2011 Wiley Periodicals, Inc.

The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

ERIC Educational Resources Information Center

Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

2017-01-01

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomkinson, B.; Wernstedt, C.; Hellman, U.

1987-11-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residuesmore » 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.« less

Effects of soybean Kunitz trypsin inhibitor on the cotton boll weevil (Anthonomus grandis).

PubMed

Franco, Octávio L; Dias, Simoni C; Magalhães, Claudio P; Monteiro, Ana C S; Bloch, Carlos; Melo, Francislete R; Oliveira-Neto, Osmundo B; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima

2004-01-01

The cotton boll weevil, Anthonomus grandis, is an economically important pest of cotton in tropical and subtropical areas of several countries in the Americas, causing severe losses due to their damage in cotton floral buds. Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities. Furthermore, in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Previously, in vivo effects of black-eyed pea trypsin chymotrypsin inhibitor (BTCI) have been demonstrated towards the boll weevil pest. Here, when neonate larvae were reared on an artificial diet containing SKTI at three different concentrations, a reduction of larval weight of up to 64% was observed for highest SKTI concentration 500 microM. The presence of SKTI caused an increase in mortality and severe deformities of larvae, pupae and adult insects. This work therefore represents the first observation of a Kunitz trypsin inhibitor active in vivo and in vitro against A. grandis. Bioassays suggested that SKTI could be used as a tool in engineering crop plants, which might exhibit increased resistance against cotton boll weevil.

Genes Expressed During Fruiting Body Formation of Agrocybe cylindracea

PubMed Central

Shim, Sung Mi; Kim, Sang Beom; Kim, Hey Young; Rho, Hyun-Su; Lee, Hyun Sook; Lee, Min Woong; Lee, U Youn; Im, Kyung Hoan

2006-01-01

Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pri1 and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research. PMID:24039501

Partial purification and identification of a metalloproteinase with anticoagulant activity from Rhizostoma pulmo (Barrel Jellyfish).

PubMed

Rastogi, Akriti; Sarkar, Angshuman; Chakrabarty, Dibakar

2017-06-15

Rhizostoma pulmo (Barrel Jellyfish) is one of the commonly found jellyfishes on the South-Goan coast of India. Here we present characterization of R. pulmo tentacle extract. The tentacle extracts were found to be capable of affecting the hemostatic system at three different levels, as it exhibited fibrinogenolysis, fibrinolysis and inhibition of ADP induced platelet aggregation. It preferentially cleaved the Aα chain of fibrinogen, followed by the Bβ chain and the γ chain. The tentacle extract also showed significant hemolytic activity against human RBCs and strong proteolytic activity for substrates like (azo) casein and gelatin. However, this proteolytic activity was completely inhibited by EDTA (metalloproteinase inhibitor) but not by PMSF (serine proteinase inhibitor). The extract was devoid of phospholipase activity. A semi-purified protein possessing fibrinogenolytic activity was obtained by a combination of ammonium sulphate precipitation and size exclusion HPLC. Atomic absorption analysis of this protein indicated presence of Zn 2+ and treatment with metalloproteinase inhibitor caused complete loss of activity. A 95 kDa metalloproteinase was identified in this fraction and was named Rhizoprotease. Protein Mass Fingerprinting of Rhizoprotease indicates it to be a novel protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

An osteoblast-derived proteinase controls tumor cell survival via TGF-beta activation in the bone microenvironment.

PubMed

Thiolloy, Sophie; Edwards, James R; Fingleton, Barbara; Rifkin, Daniel B; Matrisian, Lynn M; Lynch, Conor C

2012-01-01

Breast to bone metastases frequently induce a "vicious cycle" in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Collectively, these studies identify a novel "mini-vicious cycle" between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases.

What killed Karl Patterson Schmidt? Combined venom gland transcriptomic, venomic and antivenomic analysis of the South African green tree snake (the boomslang), Dispholidus typus.

PubMed

Pla, Davinia; Sanz, Libia; Whiteley, Gareth; Wagstaff, Simon C; Harrison, Robert A; Casewell, Nicholas R; Calvete, Juan J

2017-04-01

Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A 2 (PLA 2 ); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution*

PubMed Central

Lilla, Jennifer N.; Joshi, Ravi V.; Craik, Charles S.; Werb, Zena

2009-01-01

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. PMID:19297327

The Aged Microenvironment Influences Prostate Carcinogenesis

DTIC Science & Technology

2008-12-01

binding protein-like +36 nucleic acid binding Serpinb5 serine (or cysteine) peptidase inhibitor, clade +9 serine-type endopeptidase inhibitor activity...synthase ( phosphatidate +1.9 phosphatidate cytidylyltransferase activity Car1 carbonic anhydrase 1 +1.9 carbonate dehydratase activity;zinc ion...activity Wdr45l Wdr45 like +1.7 acid phosphatase activity;molecular_function unknown Perp PERP, TP53 apoptosis effector +1.7 structural constituent of

Characterization of a chimeric foot-and-mouth disease virus bearing bovine rhinitis B virus leader proteinase

USDA-ARS?s Scientific Manuscript database

Our recent study has shown that bovine rhinovirus type 2 (BRV2), a new member of the Aphthovirus genus, shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). Despite low sequence conservation (36percent amino acid identity) and N- and C-terminus folding differences,...

Nicotine Uses Neuron-Glia Communication to Enhance Hippocampal Synaptic Transmission and Long-term Memory

PubMed Central

López-Hidalgo, Mónica; Salgado-Puga, Karla; Alvarado-Martínez, Reynaldo; Medina, Andrea Cristina; Prado-Alcalá, Roberto A.; García-Colunga, Jesús

2012-01-01

Nicotine enhances synaptic transmission and facilitates long-term memory. Now it is known that bi-directional glia-neuron interactions play important roles in the physiology of the brain. However, the involvement of glial cells in the effects of nicotine has not been considered until now. In particular, the gliotransmitter D-serine, an endogenous co-agonist of NMDA receptors, enables different types of synaptic plasticity and memory in the hippocampus. Here, we report that hippocampal long-term synaptic plasticity induced by nicotine was annulled by an enzyme that degrades endogenous D-serine, or by an NMDA receptor antagonist that acts at the D-serine binding site. Accordingly, both effects of nicotine: the enhancement of synaptic transmission and facilitation of long-term memory were eliminated by impairing glial cells with fluoroacetate, and were restored with exogenous D-serine. Together, these results show that glial D-serine is essential for the long-term effects of nicotine on synaptic plasticity and memory, and they highlight the roles of glial cells as key participants in brain functions. PMID:23185511

Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

PubMed

Shan, Hongying; Pasin, Fabio; Tzanetakis, Ioannis E; Simón-Mateo, Carmen; García, Juan Antonio; Rodamilans, Bernardo

2018-06-01

The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Wound and methyl jasmonate induced pigeon pea defensive proteinase inhibitor has potency to inhibit insect digestive proteinases.

PubMed

Lomate, Purushottam R; Hivrale, Vandana K

2012-08-01

Wounding of plants by chewing insects or other damage induces the synthesis of defensive proteinase inhibitors (PI) in both wounded and distal unwounded leaves. In the present paper we report the characterization of inducible defensive PI from pigeon pea (Cajanus cajan) and its in vitro interaction with Helicoverpa armigera gut proteinases (HGP). We found that PI activity was induced in local as well as systemic leaves of pigeon pea by the wounding and methyl jasmonate (MeJA) application. Consistent induction of PI was observed in two wild cultivars of pigeon pea at various growth stages. The estimated molecular weight of inducible PI was ~16.5 kDa. Electrophoretic analysis and enzyme assays revealed that the induced PI significantly inhibited total gut proteinase as well as trypsin-like activity from the midgut of H. armigera. The induced PI was found to be inhibitor of trypsin as well as chymotrypsin. Study could be important to know the further roles of defensive PIs. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

PubMed

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.

PubMed

Visessanguan, Wonnop; Benjakul, Soottawat; An, Haejung

2003-03-01

A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.

Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

PubMed

Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

2016-02-19

Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

PubMed

Horio, Mao; Ishima, Tamaki; Fujita, Yuko; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji

2013-05-01

d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium

PubMed Central

Sun, Tong; Kim, Bohye; Kim, Lou W.

2013-01-01

Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3− cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3− cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. PMID:24102085

Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization.

PubMed

Portela, Maristela B; Souza, Ivete P R; Abreu, Celina M; Bertolini, Martinna; Holandino, Carla; Alviano, Celuta S; Santos, André L S; Soares, Rosangela M A

2010-11-01

  There are several kinds of oral soft tissue lesions that are common manifestations observed in human immunodeficiency virus (HIV)-infected children; for example, linear gingival erythema (LGE) that is a distinctive fiery red band along the margin of the gingivae. The etiology and pathogenesis of LGE are questionable, but a candidal origin has been suggested. Proteases are key virulence attributes produced by a variety of pathogenic fungi, including Candida. The objective of the present study is to identify the protease production in Candida species including, C. albicans (n=5), C. dubliniensis (n=1) and C. tropicalis (n=1), isolated directly from typical LGE lesions observed in six HIV-positive children, and also to test the effect of a serine protease inhibitor on the interaction of Candida spp. and epithelial cells in vitro. The ability of Candida strains to release proteases in the culture supernatant fluids was visualized by gelatin-SDS-PAGE. Gel strips containing 30-fold concentrated supernatant (1.5×10(8) yeasts) were incubated at 37°C for 48 h in 50 mM sodium phosphate buffer, pH 5.5. The concentrated supernatants were also incubated with fibronectin, laminin, immunoglobulin G, bovine serum albumin and human serum albumin. The effect of serine protease inhibitor on the interaction of Candida spp. and epithelial cells (MA 104) was measured after pre-treatment of fungi with the inhibitor (phenylmethylsulphonyl fluoride, PMSF). All the extracellular proteases were completely inhibited by PMSF, identifying these activities as serine-type proteases. Interestingly, a common 62-kDa serine protease was observed in all Candida strains. The culture supernatants, rich in serine protease activities, cleaved several soluble proteinaceous substrates. Additionally, we demonstrated that pre-treatment of C. albicans, C. dubliniensis and C. tropicalis with PMSF diminished the interaction with epithelial cells. Collectively, our results show that Candida spp. isolated from LGE lesions produced and secreted serine proteases and these enzymes may be involved in the initial colonization events. © 2010 John Wiley & Sons A/S.

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi.

PubMed

Chagas, J R; Authie, E; Serveau, C; Lalmanach, G; Juliano, L; Gauthier, F

1997-09-01

Congopain and cruzipain, the major cysteine proteinases from Trypanosoma congolense and Trypanosoma cruzi, were compared for their activities towards a series of new, sensitive fluorogenic substrates of the papain family of cysteine proteinases and for their sensitivity to inhibition by cystatins and related biotinylated peptidyl diazomethanes. Low Ki values, in the 10 pM range, were found for the interaction of both proteinases with natural cystatin inhibitors. The kinetic constants for the hydrolysis of cystatin-derived substrates, and the inhibition by related diazomethanes were essentially identical. Unlike cathepsins B and L, the related mammal papain family proteinases, congopain and cruzipain accomodate a prolyl residue in P2'. Substrates having the sequence VGGP from P2 to P2' were hydrolysed by both congopain and cruzipain with a k(cat)/Km greater than 4.10(3) mM(-1) s(-1). Irreversible diazomethane inhibitors, deduced from the unprime sequence of cystatin-derived substrates, inhibited the two parasite proteinases. N-terminal labelling of diazomethanes with a biotin group did not alter the rate of inhibition significantly, which provides a useful tool for examining the distribution of these enzymes in the parasite and in the host. Despite their similar activities on cystatin-derived substrates, congopain and cruzipain had significantly different pH-activity profiles when assayed with a cystatin-derived substrate. They were correlated with structural differences, especially at the presumed S2 subsites.

Relationship between transmembrane serine protease expression and prognosis of esophageal squamous cell carcinoma.

PubMed

Liu, G T; Shen, C; Ren, X H; Yang, L; Yu, Y M; Xiu, Y X; Li, R H; Jiang, L; Zhang, C L; Li, Y W

2017-01-01

Esophageal squamous cell carcinoma is the most common type of esophageal cancer in Eastern Europe and Asia, being the 6th most common cause of cancer deaths worldwide. The aim of this study was to analyze the expression of transmembrane serine protein in esophageal squamous cell carcinoma, and to correlate it with the clinical biological features of esophageal cancer. The expression of transmembrane protease serine 4 (TMPRSS4) mRNA and protein in carcinoma tissues and corresponding adjacent tissues and non-tumorous esophageal tissues was determined using PCR (qRT-PCR). The results show that both TMPRSS4 mRNA and protein expression were remarkably lower in adjacent normal tissues than in tumorous tissues. TMPRSS4 protein expression in esophageal carcinoma was correlated with patient demographic characteristics, tumor type, high TNM stages and overall survival (OS). Based on the experimental results, we conclude that TMPRSS4 is closely related to the occurrence, development and metastasis of esophageal squamous cell carcinoma.

RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro†

PubMed Central

Valli, Adrian; Martín-Hernández, Ana Montserrat; López-Moya, Juan José; García, Juan Antonio

2006-01-01

The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family. PMID:17005683

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

PubMed

Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2013-03-01

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy. Georg Thieme Verlag KG Stuttgart · New York.

RECK-Mediated β1-Integrin Regulation by TGF-β1 Is Critical for Wound Contraction in Mice.

PubMed

Gutiérrez, Jaime; Droppelmann, Cristian A; Contreras, Osvaldo; Takahashi, Chiaki; Brandan, Enrique

2015-01-01

Fibroblasts are critical for wound contraction; a pivotal step in wound healing. They produce and modify the extracellular matrix (ECM) required for the proper tissue remodeling. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a key regulator of ECM homeostasis and turnover. However, its role in wound contraction is presently unknown. Here we describe that Transforming growth factor type β1 (TGF-β1), one of the main pro-fibrotic wound-healing promoting factors, decreases RECK expression in fibroblasts through the Smad and JNK dependent pathways. This TGF-β1 dependent downregulation of RECK occurs with the concomitant increase of β1-integrin, which is required for fibroblasts adhesion and wound contraction through the activation of focal adhesion kinase (FAK). Loss and gain RECK expression experiments performed in different types of fibroblasts indicate that RECK downregulation mediates TGF-β1 dependent β1-integrin expression. Also, reduced levels of RECK potentiate TGF-β1 effects over fibroblasts FAK-dependent contraction, without affecting its cognate signaling. The above results were confirmed on fibroblasts derived from the Reck+/- mice compared to wild type-derived fibroblasts. We observed that Reck+/- mice heal dermal wounds more efficiently than wild type mice. Our results reveal a critical role for RECK in skin wound contraction as a key mediator in the axis: TGF-β1-RECK-β1-integrin.

Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR.

PubMed

Frank, T S; Svoboda-Newman, S M; Hsi, E D

1996-09-01

DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326.

PubMed Central

Lampel, J S; Aphale, J S; Lampel, K A; Strohl, W R

1992-01-01

The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pIJ702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase. Images PMID:1569011

The entomopathogenic fungus Metarhizium robertsii communicates with the insect host Galleria mellonella during infection.

PubMed

Mukherjee, Krishnendu; Vilcinskas, Andreas

2018-01-01

Parasitic fungi are the only pathogens that can infect insect hosts directly through their proteinaceous exoskeleton. Penetration of the cuticle requires the release of fungal enzymes, including proteinases, which act as virulence factors. Insects can sense fungal infections and activate innate immune responses, including the synthesis of antifungal peptides and proteinase inhibitors that neutralize the incoming proteinases. This well-studied host response is epigenetically regulated by histone acetylation/deacetylation. Here we show that entomopathogenic fungi can in turn sense the presence of insect-derived antifungal peptides and proteinase inhibitors, and respond by inducing the synthesis of chymotrypsin-like proteinases and metalloproteinases that degrade the host-derived defense molecules. The rapidity of this response is dependent on the virulence of the fungal strain. We confirmed the specificity of the pathogen response to host-derived defense molecules by LC/MS and RT-PCR analysis, and correlated this process with the epigenetic regulation of histone acetylation/deacetylation. This cascade of responses reveals that the coevolution of pathogens and hosts can involve a complex series of attacks and counterattacks based on communication between the invading fungal pathogen and its insect host. The resolution of this process determines whether or not pathogenesis is successful.

PRSS1 and SPINK1 mutations in idiopathic chronic and recurrent acute pancreatitis.

PubMed

Pelaez-Luna, Mario; Robles-Diaz, Guillermo; Canizales-Quinteros, Samuel; Tusié-Luna, Maria T

2014-09-07

To identify gene mutations in PRSS1 and SPINK1 in individuals with early onset idiopathic chronic or recurrent acute pancreatitis. The cationic trypsinogen gene (PRSS1; exons 2 and 3) and the serine protease inhibitor Kazal 1 gene (SPINK1; exon 3) were selectively amplified and sequenced from blood samples of 19 patients admitted to the Pancreas Clinic at our institution with chronic pancreatitis and/or idiopathic recurrent acute pancreatitis that were diagnosed or with onset before age 35. Fifty healthy volunteers served as controls. Whole blood samples were collected and gene specific sequences were amplified by polymerase chain reaction (PCR). All PCR products were subsequently sequenced in order to identify the presence of any mutations. Nineteen patients with pancreatitis (14 males; median age 24 years, range 15-48 years) were included in this study, of which five showed the presence of gene mutations. Direct sequencing results indicated the presence of two previously unidentified mutations in exon 2 of PRSS1 (V39E and N42S) in two patients with recurrent acute pancreatitis. Two cases had the N34S SPINK1 mutation. Analysis of the relatives of one patient homozygous for this mutation showed that five of the six family members carried the N34S SPINK1 mutation. Of these members, three were healthy heterozygous carriers and two were homozygotes (one sibling had diabetes, the other was healthy). Another patient was heterozygous for a novel SPINK1 mutation located on exon 3 (V46D). All members from this patient's family had normal genotypes, indicating that it was a de novo mutation. No mutations in either gene were present in the control subjects. Two novel PRSS1 mutations and one novel SPINK1 mutation were identified in Mexican patients with early onset idiopathic recurrent acute pancreatitis.

In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

PubMed

Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

2000-08-01

The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

tRNAs as Therapeutic Agents of Breast Cancer

DTIC Science & Technology

2013-07-01

their anticodon sequence. Wild-type tRNA reads codon for serine, Suppressor (Sup) tRNA for amber stop, and killer tRNA for isoleucine . Figure 6...endoplasmic reticulum (ER) is a eukaryotic organelle that performs the major functions of synthesizing and packaging pro- teins. Overloading of...anticodons tested in HeLa, tRNASer with the AAU anticodon (tRNASer(AAU)) leads to the substitution of isoleucine with serine within the proteome and

Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.

PubMed

Droux, M; Ruffet, M L; Douce, R; Job, D

1998-07-01

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.

Mutations in SLC1A4, encoding the brain serine transporter, are associated with developmental delay, microcephaly and hypomyelination.

PubMed

Damseh, Nadirah; Simonin, Alexandre; Jalas, Chaim; Picoraro, Joseph A; Shaag, Avraham; Cho, Megan T; Yaacov, Barak; Neidich, Julie; Al-Ashhab, Motee; Juusola, Jane; Bale, Sherri; Telegrafi, Aida; Retterer, Kyle; Pappas, John G; Moran, Ellen; Cappell, Joshua; Anyane Yeboa, Kwame; Abu-Libdeh, Bassam; Hediger, Matthias A; Chung, Wendy K; Elpeleg, Orly; Edvardson, Simon

2015-08-01

L-serine plays an essential role in neuronal development and function. Although a non-essential amino acid, L-serine must be synthesised within the brain because of its poor permeability by the blood-brain barrier. Within the brain, its synthesis is confined to astrocytes, and its shuttle to neuronal cells is performed by a dedicated neutral amino acid transporter, ASCT1. Using exome analysis we identified the recessive mutations, p.E256K, p.L315fs, and p.R457W, in SLC1A4, the gene encoding ASCT1, in patients with developmental delay, microcephaly and hypomyelination; seizure disorder was variably present. When expressed in a heterologous system, the mutations did not affect the protein level at the plasma membrane but abolished or markedly reduced L-serine transport for p.R457W and p.E256K mutations, respectively. Interestingly, p.E256K mutation displayed a lower L-serine and alanine affinity but the same substrate selectivity as wild-type ASCT1. The clinical phenotype of ASCT1 deficiency is reminiscent of defects in L-serine biosynthesis. The data underscore that ASCT1 is essential in brain serine transport. The SLC1A4 p.E256K mutation has a carrier frequency of 0.7% in the Ashkenazi-Jewish population and should be added to the carrier screening panel in this community. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis

PubMed Central

Hernández, Hilda M.; Marcet, Ricardo; Sarracent, Jorge

2014-01-01

Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

Classification of microbial α-amylases for food manufacturing using proteinase digestion.

PubMed

Akiyama, Takumi; Yamazaki, Takeshi; Tada, Atsuko; Ito, Yusai; Otsuki, Noriko; Akiyama, Hiroshi

2014-09-01

Enzymes produced by microorganisms and plants are used as food additives to aid the processing of foods. Identification of the origin of these enzyme products is important for their proper use. Proteinase digestion of α-amylase products, followed by high performance liquid chromatography (HPLC) analysis, was applied to α-amylase from the mold Aspergillus species, the bacteria Bacillus species, and the actinomycetes Saccharomonospora species. Eighteen commercial products of α-amylase were digested with trypsin and endoproteinase Lys-C and HPLC analyzed. For some proteinase/sample combinations, the area of the intact α-amylase peak decreased and new peaks were detected after digestion. The presence and retention times of the novel peaks were used to group the products. The results from this method, called the proteinase digestion-HPLC method, allowed the classification of the α-amylase products into 10 groups, whereas the results from sodium dodecyl sulfate polyacrylamide gel electrophoresis allowed their classification into seven groups.

[ELISA in the determination of antiphosphatidylserine antibodies].

PubMed

Fialová, L; Mikulíková, L; Fisar, Z; Beitlová, D; Malbohan, I

1996-05-01

Antiphospholipid antibodies (APA) are a group of antibodies against various phospholipid antigens. In order to extend the spectrum of examined specificities of antiphospholipid antibodies the authors elaborated an ELISA method for assessment of antiphosphatidyl serine antibodies (APSA). As antigen they used phosphatidyl serine isolated from the white matter of cattle brain. The ELISA method was tested by examining APSA in 12 patients with rheumatic diseases, 24 women with reproductive disorders and 50 patients with testicular tumours and the results were compared with examinations of anticardiolipin antibodies. The concurrent presence of both types of antibodies was recorded in 20.8% women with reproductive disorders and in 14% of the patients with testicular tumours. In these groups antiphosphatidyl serine antibodies were found more frequently.

Serine is the major residue for ADP-ribosylation upon DNA damage

PubMed Central

Dauben, Helen

2018-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling. PMID:29480802

Modeling extracellular matrix degradation balance with proteinase/transglutaminase cycle.

PubMed

Larreta-Garde, Veronique; Berry, Hugues

2002-07-07

Extracellular matrix mass balance is implied in many physiological and pathological events, such as metastasis dissemination. Widely studied, its destructive part is mainly catalysed by extracellular proteinases. Conversely, the properties of the constructive part are less obvious, cellular neo-synthesis being usually considered as its only element. In this paper, we introduce the action of transglutaminase in a mathematical model for extracellular matrix remodeling. This extracellular enzyme, catalysing intermolecular protein cross-linking, is considered here as a reverse proteinase as far as the extracellular matrix physical state is concerned. The model is based on a proteinase/transglutaminase cycle interconverting insoluble matrix and soluble proteolysis fragments, with regulation of cellular proteinase expression by the fragments. Under "closed" (batch) conditions, i.e. neglecting matrix influx and fragment efflux from the system, the model is bistable, with reversible hysteresis. Extracellular matrix proteins concentration abruptly switches from low to high levels when transglutaminase activity exceeds a threshold value. Proteinase concentration usually follows the reverse complementary kinetics, but can become apparently uncoupled from extracellular matrix concentration for some parameter values. When matrix production by the cells and fragment degradation are taken into account, the dynamics change to sustained oscillations because of the emergence of a stable limit cycle. Transitions out of and into oscillation areas are controlled by the model parameters. Biological interpretation indicates that these oscillations could represent the normal homeostatic situation, whereas the other exhibited dynamics can be related to pathologies such as tumor invasion or fibrosis. These results allow to discuss the insights that the model could contribute to the comprehension of these complex biological events.

Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli.

PubMed

Prüss, B M

1998-09-01

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Properties of a Kunitz-type trypsin inhibitor from Delonix regia seeds against digestive proteinases of Anagasta kuehniella (Z.) and Corcyra cephalonica (S.) (Lepidoptera: Pyralidae).

PubMed

Macedo, M L R; Pando, S C; Chevreuil, L R; Marangoni, S

2009-01-01

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

PubMed

Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

2016-06-23

To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

Beta Catenin in Prostate Cancer Apoptosis

DTIC Science & Technology

2014-04-01

indicate that, Glycogen Synthase Kinase 3β (GSK3β) might be a key player in mediating this. GSK3β, a multifunctional serine/ threonine kinase regulates...for TRAIL-TZD-induced apoptosis in prostate cancer cells. AMPK is a family of serine/ threonine protein kinase and is highly conserved from yeast to...metabolic syndrome and Type 2 diabetes . We used C42-DN (stably overexpressing AMPK α1-dominant negative) and C42-EV (empty vector) prostate cancer cell

Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

1988-06-01

E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

PubMed

Price, Joseph A

2015-09-01

Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human endometrial matrix metalloproteinase-2, a putative menstrual proteinase. Hormonal regulation in cultured stromal cells and messenger RNA expression during the menstrual cycle.

PubMed Central

Irwin, J C; Kirk, D; Gwatkin, R B; Navre, M; Cannon, P; Giudice, L C

1996-01-01

Proteinases are likely effectors of endometrial menstrual breakdown. We have investigated proteinase production by human endometrial stromal cells subjected in vitro to progesterone (P) withdrawal, the physiologic stimulus for menstruation. Culture media of cells exposed to estradiol, P, or estradiol plus P had low levels of proteolytic activity similar to cultures maintained in the absence of steroids. P withdrawal, or addition of RU486 to P-treated cultures, stimulated proteinase secretion. The stromal cell proteinase was characterized by gelatin zymography, inhibitor profile, and organomercurial activation, as a metalloproteinase present mostly as a 66-kD proenzyme with lower levels of a 62-kD active form. The P withdrawal-induced metalloproteinase was identified as matrix metalloproteinase-2 (MMP-2) by Western blotting. The increase of MMP-2 induced by P withdrawal was associated with the metalloproteinase-dependent breakdown of stromal cultures, involving dissolution of extracellular matrix and dissociation of stromal cells. Northern analysis showed the differential expression of MMP-2 mRNA in late secretory phase endometrium. These findings are consistent with the involvement of stromal cell-derived MMP-2 in the proteolysis of extracellular matrix promoting cyclic endometrial breakdown and the onset of menstrual bleeding. PMID:8567965

Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

PubMed Central

Simon, Stéphanie; Nugier, Jérôme; Morel, Nathalie; Boutal, Hervé; Créminon, Christophe; Benestad, Sylvie L.; Andréoletti, Olivier; Lantier, Frédéric; Bilheude, Jean-Marc; Feyssaguet, Muriel; Biacabe, Anne-Gaëlle; Baron, Thierry

2008-01-01

The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)–infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc. PMID:18394279

TIME management by medicinal larvae.

PubMed

Pritchard, David I; Čeřovský, Václav; Nigam, Yamni; Pickles, Samantha F; Cazander, Gwendolyn; Nibbering, Peter H; Bültemann, Anke; Jung, Wilhelm

2016-08-01

Wound bed preparation (WBP) is an integral part of the care programme for chronic wounds. The acronym TIME is used in the context of WBP and describes four barriers to healing in chronic wounds; namely, dead Tissue, Infection and inflammation, Moisture imbalance and a non-migrating Edge. Larval debridement therapy (LDT) stems from observations that larvae of the blowfly Lucilia sericata clean wounds of debris. Subsequent clinical studies have proven debriding efficacy, which is likely to occur as a result of enzymatically active alimentary products released by the insect. The antimicrobial, anti-inflammatory and wound healing activities of LDT have also been investigated, predominantly in a pre-clinical context. This review summarises the findings of investigations into the molecular mechanisms of LDT and places these in context with the clinical concept of WBP and TIME. It is clear from these findings that biotherapy with L. sericata conforms with TIME, through the enzymatic removal of dead tissue and its associated biofilm, coupled with the secretion of defined antimicrobial peptides. This biotherapeutic impact on the wound serves to reduce inflammation, with an associated capacity for an indirect effect on moisture imbalance. Furthermore, larval serine proteinases have the capacity to alter fibroblast behaviour in a manner conducive to the formation of granulation tissue. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis

PubMed Central

Sales, Katiuchia Uzzun; Friis, Stine; Konkel, Joanne E.; Godiksen, Sine; Hatakeyama, Marcia; Hansen, Karina K.; Rogatto, Silvia Regina; Szabo, Roman; Vogel, Lotte K.; Chen, Wanjun; Gutkind, J. Silvio; Bugge, Thomas H.

2014-01-01

The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of NFκB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia, and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis. PMID:24469043

Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis.

PubMed

Sales, K U; Friis, S; Konkel, J E; Godiksen, S; Hatakeyama, M; Hansen, K K; Rogatto, S R; Szabo, R; Vogel, L K; Chen, W; Gutkind, J S; Bugge, T H

2015-01-15

The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of nuclear factor (NF)κB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis.

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins.

PubMed

del Valle, Luis J

2005-06-01

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

PubMed

Ortiz, Gustavo; Salica, Juan P; Chuluyan, Eduardo H; Gallo, Juan E

2014-11-18

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway

PubMed Central

Liu, Xiaochen; Zhang, Bin; McBride, Jeffrey D.; Zhou, Kevin; Lee, Kyungwon; Zhou, Yueping; Liu, Zuguo; Ma, Jian-xing

2013-01-01

Kallistatin is a member of the serine proteinase inhibitor superfamily. Kallistatin levels have been shown to be decreased in the vitreous while increased in the circulation of patients with diabetic retinopathy (DR). Overactivation of the Wnt pathway is known to play pathogenic roles in DR. To investigate the role of kallistatin in DR and in Wnt pathway activation, we generated kallistatin transgenic (kallistatin-TG) mice overexpressing kallistatin in multiple tissues including the retina. In the oxygen-induced retinopathy (OIR) model, kallistatin overexpression attenuated ischemia-induced retinal neovascularization. In diabetic kallistatin-TG mice, kallistatin overexpression ameliorated retinal vascular leakage, leukostasis, and overexpression of vascular endothelial growth factor and intracellular adhesion molecule. Furthermore, kallistatin overexpression also suppressed Wnt pathway activation in the retinas of the OIR and diabetic models. In diabetic Wnt reporter (BAT-gal) mice, kallistatin overexpression suppressed retinal Wnt reporter activity. In cultured retinal cells, kallistatin blocked Wnt pathway activation induced by high glucose and by Wnt ligand. Coprecipitation and ligand-binding assays both showed that kallistatin binds to a Wnt coreceptor LRP6 with high affinity (Kd = 4.5 nmol/L). These observations suggest that kallistatin is an endogenous antagonist of LRP6 and inhibitor of Wnt signaling. The blockade of Wnt signaling may represent a mechanism for its antiangiogenic and antineuroinflammatory effects. PMID:23884893

Characterization of a potent platelet aggregation inducer from Cerastes cerastes (Egyptian sand viper) venom.

PubMed

Basheer, A R; el-Asmar, M F; Soslau, G

1995-07-03

A potent, proteinaceous inducer of platelet aggregation designated as IVa, has been purified to homogeneity from Cerastes cerastes venom by molecular sieve and ion exchange chromatography. It is composed of 2 subunits with total M(r) of 62,000 as shown by native gel chromatography and chemical cross-linking with disuccinimidyl suberate. It is not clear at the present time whether both subunits are identical gene products, however, both have identical N-terminal sequences for the first 15 amino acids. The protein has a pI above 9.6. IVa (0.1 micrograms/ml) could aggregate platelets up to 80% and was inhibited by p-APMSF, leupeptin, iodoacetamide, protein kinase C inhibitor, phosphatase inhibitor, ATP and PGE1, while it was insensitive to acetylsalicylic acid, ADP scavenger system, protein kinase A inhibitor and hirudin. Protein IVa is a serine proteinase with thrombin-like activity as it hydrolysed thrombin chromogenic substrate CBS 34.47, its aggregatory activity was partially inhibited by monoclonal antibodies against GPIb and the thrombin receptor, as was the thrombin, and its ability to induce intracellular Ca2+ release was blocked by pretreating platelets with thrombin. Unlike thrombin, the IVa protein showed very weak coagulant activity as indicated by plasma recalcification time and fibrinogen clotting time although it could hydrolyse fibrinogen alpha-chains.

RNA-Seq Analyses for Two Silkworm Strains Reveals Insight into Their Susceptibility and Resistance to Beauveria bassiana Infection.

PubMed

Xing, Dongxu; Yang, Qiong; Jiang, Liang; Li, Qingrong; Xiao, Yang; Ye, Mingqiang; Xia, Qingyou

2017-02-10

The silkworm Bombyx mori is an economically important species. White muscardine caused by Beauveria bassiana is the main fungal disease in sericulture, and understanding the silkworm responses to B. bassiana infection is of particular interest. Herein, we investigated the molecular mechanisms underlying these responses in two silkworm strains Haoyue (HY, sensitive to B. bassiana ) and Kang 8 (K8, resistant to B. bassiana ) using an RNA-seq approach. For each strain, three biological replicates for immersion treatment, two replicates for injection treatment and three untreated controls were collected to generate 16 libraries for sequencing. Differentially expressed genes (DEGs) between treated samples and untreated controls, and between the two silkworm strains, were identified. DEGs and the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the two strains exhibited an obvious difference. Several genes encoding cuticle proteins, serine proteinase inhibitors (SPI) and antimicrobial peptides (AMP) and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. These results revealed insight into the resistance and susceptibility of two silkworm strains against B. bassiana infection and provided a roadmap for silkworm molecular breeding to enhance its resistance to B. bassiana .

RNA-Seq Analyses for Two Silkworm Strains Reveals Insight into Their Susceptibility and Resistance to Beauveria bassiana Infection

PubMed Central

Xing, Dongxu; Yang, Qiong; Jiang, Liang; Li, Qingrong; Xiao, Yang; Ye, Mingqiang; Xia, Qingyou

2017-01-01

The silkworm Bombyx mori is an economically important species. White muscardine caused by Beauveria bassiana is the main fungal disease in sericulture, and understanding the silkworm responses to B. bassiana infection is of particular interest. Herein, we investigated the molecular mechanisms underlying these responses in two silkworm strains Haoyue (HY, sensitive to B. bassiana) and Kang 8 (K8, resistant to B. bassiana) using an RNA-seq approach. For each strain, three biological replicates for immersion treatment, two replicates for injection treatment and three untreated controls were collected to generate 16 libraries for sequencing. Differentially expressed genes (DEGs) between treated samples and untreated controls, and between the two silkworm strains, were identified. DEGs and the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the two strains exhibited an obvious difference. Several genes encoding cuticle proteins, serine proteinase inhibitors (SPI) and antimicrobial peptides (AMP) and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. These results revealed insight into the resistance and susceptibility of two silkworm strains against B. bassiana infection and provided a roadmap for silkworm molecular breeding to enhance its resistance to B. bassiana. PMID:28208575

Mitochondria are an early target of oxidative modifications in senescing legume nodules.

PubMed

Matamoros, Manuel A; Fernández-García, Nieves; Wienkoop, Stefanie; Loscos, Jorge; Saiz, Ana; Becana, Manuel

2013-02-01

Legume nodule senescence is a poorly understood process involving a decrease in N(2) fixation and an increase in proteolytic activity. Some physiological changes during nodule aging have been reported, but scarce information is available at the subcellular level. Biochemical, immunological and proteomic approaches were used to provide insight into the effects of aging on the mitochondria and cytosol of nodule host cells. In the mitochondria, the oxidative modification of lipids and proteins was associated with a marked decline in glutathione, a reduced capacity to regenerate ascorbate, and upregulation of alternative oxidase and manganese superoxide dismutase. In the cytosol, there were consistent reductions in the protein concentrations of carbon metabolism enzymes, inhibition of protein synthesis and increase in serine proteinase activity, disorganization of cytoskeleton, and a sharp reduction of cytosolic proteins, but no detectable accumulation of oxidized molecules. We conclude that nodule mitochondria are an early target of oxidative modifications and a likely source of redox signals. Alternative oxidase and manganese superoxide dismutase may play important roles in controlling ROS concentrations and the redox state of mitochondria. The finding that specific methionine residues of a cytosolic glutamine synthetase isoform are sulfoxidized suggests a regulatory role of this enzyme in senescing nodules. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Neutral endopeptidase inhibitor potentiates the tachykinin-induced increase in ciliary beat frequency in rabbit trachea.

PubMed

Kondo, M; Tamaoki, J; Takizawa, T

1990-08-01

We used cultured rabbit tracheal epithelium to determine the effect of mammalian-derived tachykinin on airway ciliary activity and its modulation by neutral endopeptidase EC 3.4.24.11 (NEP). Neurokinin A (NKA) caused dose-dependent increases in ciliary beat frequency (CBF), as measured by a photoelectric method, with the maximal increase from the baseline 15.7 +/- 1.7% (mean +/- SEM, p less than 0.01), whereas substance P (SP) had no effect. The NKA-induced increase in CBF was not inhibited by phentolamine, propranolol, or atropine, but it was abolished by the tachykinin antagonist [D-Pro2, D-Trp7,9]SP. Pretreatment of tissue with thiorphan (10(-5) M), a NEP inhibitor, had little effect on CBF responses to NKA; however, it significantly potentiated the responses to SP (14.9 +/- 3.0%, p less than 0.01). Other peptidase inhibitors, including captopril, bestatin, and leupeptin, did not alter the tachykinin-induced CBF response, suggesting that angiotensin converting enzyme, aminopeptidases, and serine proteinases do not modulate ciliary activity in response to tachykinins. These results suggest that NKA increases CBF by acting directly on tachykinin receptors and that NEP may play a role in modulating the tachykinin-induced stimulatory effects on CBF.

Human uterus myoma and gene expression profiling: A novel in vitro model for studying secretory leukocyte protease inhibitor-mediated tumor invasion.

PubMed

Mikami, Yoshikazu; Fukushima, Atsushi; Komiyama, Yusuke; Iwase, Takashi; Tsuda, Hiromasa; Higuchi, Yasuhiko; Hayakawa, Satoshi; Kuyama, Kayo; Komiyama, Kazuo

2016-08-28

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that diminishes tissue destruction during inflammation. A recent report revealed high levels of SLPI expression in the oral carcinoma cell. In addition, overexpression of SLPI up-regulates metastasis in lung carcinoma cells. On the other hand, matrix metalloproteinases (MMPs) are proteinases that participate in extracellular matrix degradation. SLPI and MMPs are involved as accelerators of the tumor invasion process; however, their exact roles are not fully understood. Understanding the mechanism of tumor invasion requires models that take the effect of microenvironmental factors into account. In one such in vitro model, different carcinoma cells have been shown to invade myoma tissue in highly distinct patterns. We have used this myoma model, as it provides a more natural stroma-like environment, to investigate the role of SLPI in tumor invasion. Our results indicate that the model provides a relevant matrix for tumor invasion studies, and that SLPI is important for the invasion of oral carcinoma Ca9-22 cells in conjunction with MMPs. Furthermore, using bioinformatics analysis, we have identified candidates as key molecules involved in SLPI-mediated tumor invasion. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

PubMed Central

Sanz, Yolanda; Toldrá, Fidel

2001-01-01

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu2+, Hg2+, and Zn2+). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. Km s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed. PMID:11282638

Negative role of RIG-I serine 8 phosphorylation in the regulation of interferon-beta production.

PubMed

Nistal-Villán, Estanislao; Gack, Michaela U; Martínez-Delgado, Gustavo; Maharaj, Natalya P; Inn, Kyung-Soo; Yang, Heyi; Wang, Rong; Aggarwal, Aneel K; Jung, Jae U; García-Sastre, Adolfo

2010-06-25

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

An Osteoblast-Derived Proteinase Controls Tumor Cell Survival via TGF-beta Activation in the Bone Microenvironment

PubMed Central

Thiolloy, Sophie; Edwards, James R.; Fingleton, Barbara; Rifkin, Daniel B.; Matrisian, Lynn M.; Lynch, Conor C.

2012-01-01

Background Breast to bone metastases frequently induce a “vicious cycle” in which osteoclast mediated bone resorption and proteolysis results in the release of bone matrix sequestered factors that drive tumor growth. While osteoclasts express numerous proteinases, analysis of human breast to bone metastases unexpectedly revealed that bone forming osteoblasts were consistently positive for the proteinase, MMP-2. Given the role of MMP-2 in extracellular matrix degradation and growth factor/cytokine processing, we tested whether osteoblast derived MMP-2 contributed to the vicious cycle of tumor progression in the bone microenvironment. Methodology/Principal Findings To test our hypothesis, we utilized murine models of the osteolytic tumor-bone microenvironment in immunocompetent wild type and MMP-2 null mice. In longitudinal studies, we found that host MMP-2 significantly contributed to tumor progression in bone by protecting against apoptosis and promoting cancer cell survival (caspase-3; immunohistochemistry). Our data also indicate that host MMP-2 contributes to tumor induced osteolysis (μCT, histomorphometry). Further ex vivo/in vitro experiments with wild type and MMP-2 null osteoclast and osteoblast cultures identified that 1) the absence of MMP-2 did not have a deleterious effect on osteoclast function (cd11B isolation, osteoclast differentiation, transwell migration and dentin resorption assay); and 2) that osteoblast derived MMP-2 promoted tumor survival by regulating the bioavailability of TGFβ, a factor critical for cell-cell communication in the bone (ELISA, immunoblot assay, clonal and soft agar assays). Conclusion/Significance Collectively, these studies identify a novel “mini-vicious cycle” between the osteoblast and metastatic cancer cells that is key for initial tumor survival in the bone microenvironment. In conclusion, the findings of our study suggest that the targeted inhibition of MMP-2 and/or TGFβ would be beneficial for the treatment of bone metastases. PMID:22238668

Studies on Proteinases from Some Blood-Sucking Insects,

DTIC Science & Technology

ovinus, and Pediculus humanus, but not in those of Cimex lectularius or Rhodnius prolixus. The trypsin and chymotrypsin have been partially... Cimex and Rhodnius appear to have a high molecular weight proteinase with optimal activity at pH 5 in their midguts. (Author)

A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

PubMed Central

Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

2016-01-01

Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

Accessory cells in physiological lymphoid tissue from the intestine: an immunohistochemical study.

PubMed

Sarsfield, P; Rinne, A; Jones, D B; Johnson, P; Wright, D H

1996-03-01

We report a study of the organization of accessory cell populations, in normal mucosal lymphoid tissue from small intestine (8 cases), large intestine (6) and appendix (9) using a panel of monoclonal antibodies and polyclonal antisera in paraffin-embedded tissue. Two populations were identified in dome areas, one positive for acid cysteine proteinase inhibitor and HLA class II (WR18) only and the second positive for S-100 protein, CD68, and WR18 and negative for acid cysteine proteinase inhibitor and factor XIIIa. Superficial colonic mucosal and small intestinal villous tip macrophages stained positively with CD68 and WR18 only, while deeper cryptal and submucosal populations exhibited additional positivity for factor XIIIa, but both populations were negative for acid cysteine proteinase inhibitor and S-100 protein. Germinal centre macrophages were positive for CD68, WR18 and acid cysteine proteinase inhibitor and negative for factor XIIIa, and S-100 protein. T zone dendritic cells included a population which stained positively for S-100 protien, WR18 and were negative for factor XIIIa, CD68 and acid cysteine proteinase inhibitor, an immunophenotype typical of interdigitating dendritic reticulum cells. This distribution of phenotypically identifiable accessory cell subpopulations was apparent at all three sites examined. We suggest that the specialized subpopulations of dendritic cells staining for S-100 protein and for acid cysteine proteinase inhibitor which are restricted to the dome areas, may have a potential role in the transfer of antigen across the epithelium to the germinal centres, while factor XIIIa appears to identify a tissue macrophage population with a potential role in stromal modulation distant from direct antigen challenge.

[Detection of split products of the immunoglobulins IgG, IgA and IgM during chronic otitis media (author's transl)].

PubMed

Kastenbauer, E R; Hochgesand, K; Hochstrasser, K; Tappermann, G

1975-07-01

Proteolytic enzymes such as pepsine or papaine are able to split IgG antibodies into large fragments in vitro. These immunoglobulin fragments (IgG, IgA, IgM) were now detected in vivo from the purulent secretions of cholesteatoma, chronic otitis media and radical mastoid cavities. During chronic otitis media the intact immunoglobulins are split due to the proteolytic activity of neutral proteinases. These fragments were qualitatively and quantitatively investigated by means of various immunological procedures. After the immunoelectrophoretic separation of the purulent middle-ear-secretions and after diffusion against anti-IgG-, anti-IgA- and anti-IgM- serum double precipitate lines could be observed especially in middle-ear-secretion with a bacterial flora of pseudomonas aeruginosa (pyocyanea) and of the proteus-providencia-group. This was the first proof of the presence of split products of the immunoglobulins. The exact demonstration of these split products could be carried out by gel-filtration and fractionation of the intact and split immunoglobulins. During chronic otitis media intact immunoglobulins are split by leucocytic and extracellular bacterial proteinases into fragments of different molecular weight. The most malignant extracellular proteinases with the greatest proteolytic activity against intact immunoglobulins are the bacterial proteinases of pseudomonas aeruginosa. These proteinases can not be inhibited by the other serum proteinaseinhibitors except for alpha-2-macroglobulin of the human blood serum. This inhibitor has a very high molecular weight so that we can not find it in a higher concentration in the middle-ear-secretion. We can liberate this inhibitor by injuring the blood vessels during a tympanoplasty. In this way we get an inhibitory effect against these proteinases and combined with an appropriate antibiotic therapy we can cure a chronic otitis media.

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

PubMed

Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

2005-08-01

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

PubMed Central

Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

2012-01-01

This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.

PubMed

Wenig, Katja; Chatwell, Lorenz; von Pawel-Rammingen, Ulrich; Björck, Lars; Huber, Robert; Sondermann, Peter

2004-12-14

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.

Serum α1-proteinase inhibitor concentrations in dogs with exocrine pancreatic disease, chronic hepatitis or proteinuric chronic kidney disease.

PubMed

Heilmann, R M; Grützner, N; Hokamp, J A; Lidbury, J A; Xenoulis, P G; Suchodolski, J S; Nabity, M B; Cianciolo, R; Steiner, J M

2018-06-01

Serum canine α 1 -proteinase inhibitor (cα 1 -PI) concentrations were evaluated in dogs with pancreatitis (n=24), exocrine pancreatic insufficiency (EPI; n=29), chronic hepatitis (CH; n=11) or proteinuric chronic kidney disease (CKD-P; n=61) to determine whether systemic proteinase/proteinase-inhibitor balance is altered in these conditions. Dogs with CKD-P had significantly lower cα 1 -PI concentrations than dogs with pancreatitis, EPI or CH; 16% of dogs with CKD-P had serum cα 1 -PI concentrations below the reference interval. Serum and urine cα 1 -PI concentrations were inversely correlated in dogs with CKD-P, but not in dogs with CH. This suggests that renal loss of cα 1 -PI contributes to decreased serum concentrations in dogs with CKD-P, while hepatic cα 1 -PI synthesis with CH either is not compromised or is counterbalanced by extrahepatic production. Copyright © 2018 Elsevier Ltd. All rights reserved.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

PubMed Central

Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

2018-01-01

ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection. PMID:29437971

Oil-free hyaluronic acid matrix for serial femtosecond crystallography

NASA Astrophysics Data System (ADS)

Sugahara, Michihiro; Song, Changyong; Suzuki, Mamoru; Masuda, Tetsuya; Inoue, Shigeyuki; Nakane, Takanori; Yumoto, Fumiaki; Nango, Eriko; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Numata, Keiji; Iwata, So

2016-04-01

The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

Reprogramming the virulence: Insect defense molecules navigating the epigenetic landscape of Metarhizium robertsii.

PubMed

Hussain, Abid

2018-01-01

Metarhizium species are the leading bio-control agents well characterized regarding pathogenicity to agricultural, forest, public health, stored grains and urban insect pests. They infect the target host through the tight conidial adherence with the insect cuticle. Conidial binding to the insect cuticle drive the systematic integrated disease development events in target host to impart pathogenesis. However, there is growing evidence that virulence of the pathogen is directly related with proteolytic enzymes including metalloproteinases, chymotrypsin-like proteinases and subtilisin-like proteinases. Successful host pathogenesis is the selection of right set of virulence-related proteinases, which evolved as a result of host-pathogen coevolution.

Identification and characterization of cysteine proteinases of Trypanosoma evansi.

PubMed

Yadav, S C; Kumar, R; Kumar, S; Tatu, U; Singh, R K; Gupta, A K

2011-09-01

Trypanosoma evansi is a causative agent of 'surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

Spatial distribution of digestive proteinases in the midgut of the Pacific white shrimp (Litopenaeus vannamei) indicates the existence of endo-ectoperitrophic circulation in Crustacea.

PubMed

Alexandre, Daniel; Ozório, Renata A; Derner, Roberto B; Fracalossi, Débora M; Oliveira, Gabriel B; Samuels, Richard I; Terra, Walter R; Silva, Carlos P

2014-01-01

The effect of dietary protein concentration on the spatial distribution of digestive proteinases in the shrimp Litopenaeus vannamei indicates the existence of endo-ectoperitrophic enzyme circulation in this species. Samples recovered from the midgut gland tissues, stomach contents, three different portions of the midgut and feces were used for quantitative and qualitative analyses of the composition and distribution of the digestive proteinases. Animals were divided into three different groups: (1) animals (controls) fed with a commercial 35% protein diet, (2) animals fed with a commercial diet supplemented with ovalbumin to a final protein concentration of 60%; (3) animals fed with an 80% protein diet. Quantitative determinations using different substrates and zymograms showed that increasing protein concentration in the diet alters the distribution of proteinases along the digestive tract. Composition of proteinases in the midgut gland, stomach contents, midgut sections and feces were similar, but not identical. Chymotrypsin and trypsin paralogues were identified in all enzyme sources in a concentration gradient along the midgut in the control shrimp, the expected distribution supporting the existence of a recycling mechanism. The occurrence of a peritrophic membrane in other Decapoda suggests that endo-ectoperitrophic circulation of digestive enzymes and nutrients may also occur in other crustaceans and also extends beyond the Insecta. Copyright © 2014 Elsevier Inc. All rights reserved.

Insight into the interactions of proteinase inhibitor-alpha-2-macroglobulin with hypochlorite-thermal analysis and biophysical approach.

PubMed

Siddiqui, Tooba; Zia, Mohammad Khalid; Ali, Syed Saqib; Ahsan, Haseeb; Khan, Fahim Halim

2018-05-17

Hypochlorous acid, an active bleaching agent is one of the major oxidants produced by neutrophils under physiological conditions. It is a potent reactive oxygen species (ROS) which causes oxidation of biomolecules. Treatment of proteins with hypochlorite results in direct oxidative damage to proteins. Alpha-2-macroglobulin is a major proteinase inhibitor and it can inhibit proteinase of any kind regardless of specificity and catalytic mechanism. The proteinase-antiproteinase balance plays an important role in mediating inflammation associated tissue destruction. In this paper, we have studied hypochlorite induced modifications in proteinase inhibitor-alpha-2-macroglobulin via biophysical techniques such as absorption spectroscopy, fluorescence spectroscopy, circular dichroism (CD), fourier transform infrared spectrometery (FTIR) and isothermal titration calorimetry (ITC). It was found that hypochlorite decreases the anti-proteolytic potential and causes inactivation of sheep alpha-2-macroglobulin. It also causes structural and functional change in alpha-2-macroglobulin as evident by absorption spectroscopy and fluorescence spectroscopy. Change in secondary structure of alpha-2-macroglobulin was confirmed by CD and FTIR. Thermodynamics parameters such as entropy (ΔS), enthalpy (ΔH) and Gibb's free energy changes (ΔG). The number of binding sites (N) of alpha-2-macroglobulin-HOCl binding in solution was determined by isothermal titration calorimetry and it was found that binding of hypochlorite with alpha-2-macroglobulin was exothermic in nature. Copyright © 2017. Published by Elsevier B.V.

Nma111p, the pro-apoptotic HtrA-like nuclear serine protease in Saccharomyces cerevisiae: a short survey.

PubMed

Fahrenkrog, Birthe

2011-10-01

The baker's yeast, Saccharomyces cerevisiae, is also capable of undergoing programmed cell death or apoptosis, for example in response to viral infection as well as during chronological and replicative aging. Intrinsically, programmed cell death in yeast can be induced by, for example, H2O2, acetic acid or the mating-type pheromone. A number of evolutionarily conserved apoptosis-regulatory proteins have been identified in yeast, one of which is the HtrA (high-temperature requirement A)-like serine protease Nma111p (Nma is nuclear mediator of apoptosis). Nma111p is a nuclear serine protease of the HtrA family, which targets Bir1p, the only known inhibitor-of-apoptosis protein in yeast. Nma111p mediates apoptosis in a serine-protease-dependent manner and exhibits its activity exclusively in the nucleus. How the activity of Nma111p is regulated has remained largely elusive, but some evidence points to a control by phosphorylation. Current knowledge of Nma111p's function in apoptosis will be discussed in the present review.

Differential expression of Annexin 2, SPINK1, and Hsp60 predict progression of prostate cancer through bifurcated WHO Gleason score categories in African American men.

PubMed

Beyene, Desta A; Naab, Tammey J; Kanarek, Norma F; Apprey, Victor; Esnakula, Ashwini; Khan, Farahan A; Blackman, Marc R; Brown, Collis A; Hudson, Tamaro S

2018-08-01

Although studies have observed several markers correlate with progression of prostate cancer (PCa), no specific markers have been identified that accurately predict the progression of this disease, even in African American (AA) men who are generally at higher risk than other ethnic groups. The primary goal of this study was to explore whether three markers could predict the progression of PCa. We investigated protein expression of Annexin 2 (ANX2), serine peptidase inhibitor, kazal type 1(SPINK1)/tumor-associated trypsin inhibitor (TATI), and heat shock protein 60 (Hsp60) in 79 archival human prostate trans-rectal ultrasound (TRUS) biopsy tissues according to a modified World Health Organization (WHO) classification: normal (WHO1a), Gleason Score (GS6 (WHO1b), GS7 subgroups (WHO2 = 3 + 4, WHO3 = 4 + 3), GS8 (WHO4), and GS9-10 (WHO5). AA men aged 41-90 diagnosed from 1990 to 2013 at Howard University were included. Automated staining assessed expression of each biomarker. Spearman correlation assessed the direction and relationship between biomarkers, WHO and modified WHO GS, age, and 5-year survival. A two-tailed t-test and ANOVA evaluated biomarkers expression in relationship to WHO normal and other GS levels, and between WHO GS levels. A logistic and linear regression analysis examined the relationship between biomarker score and WHO GS categories. Kaplan-Meier curves graphed survival. ANX2 expression decreased monotonically with the progression of PCa while expression of SPINK1/TATI and Hsp60 increased but had a more WHO GS-specific effect; SPINK1/TATI differed between normal and GS 2-6 and HSP60 differed between GS 7 and GS 2-6. WHO GS was found to be significantly and negatively associated with ANX2, and positively with SPINK1/TATI and Hsp60 expression. High SPINK1/TATI expression together with the low ANX2 expression at higher GS exhibited a bi-directional relationship that is associated with PCa progression and survival. Importantly, the data reveal that ANX2, and SPINK1/TAT1 highly associate with WHO GS and with the transition from one stage of PrCa to the next in AA men. Future research is needed in biracial and larger population studies to confirm this dynamic relationship between ANX2 and SPINK1 as independent predictors of PCa progression in all men. © 2018 Wiley Periodicals, Inc.

Impaired growth and development of Colorado potato beetle larvae on potato plants overexpressing the oryzacystatin II gene

USDA-ARS?s Scientific Manuscript database

Plant proteinase inhibitors are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. Oryzacystatins I and II (OCI and OCII) show potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate ...

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

PubMed

Dandoy, Damien; Fremaux, Christophe; de Frahan, Marie Henry; Horvath, Philippe; Boyaval, Patrick; Hols, Pascal; Fontaine, Laetitia

2011-08-30

In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase.

PubMed

Rawat, Reetika; Xu, Zeng-Fu; Yao, Kwok-Ming; Chye, Mee-Len

2005-03-01

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

Bowman-Birk proteinase inhibitor from Cajanus cajan seeds: purification, characterization, and insecticidal properties.

PubMed

Prasad, Elaprolu R; Merzendorfer, H; Madhurarekha, C; Dutta-Gupta, A; Padmasree, K

2010-03-10

A red gram proteinase inhibitor (RgPI) was purified from red gram ( Cajanus cajan ) seeds by using ammonium sulfate precipitation and ion-exchange, affinity, and gel filtration chromatography. SDS-PAGE under nonreducing condition revealed two protein bands with molecular masses of approximately 8.5 and approximately 16.5 kDa corresponding to monomeric and dimeric forms of RgPI, respectively. Similarly, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry also confirmed the presence of dimer as well as other oligomeric forms: trimer, tetramer, and pentamer. Reduction of RgPI with dithiothreitol (DTT) led to the dissociation of the dimeric and oligomeric forms. Native-PAGE and two-dimensional gel electrophoresis indicated the existence of isoinhibitors with pI values of 5.95, 6.25, 6.50, 6.90, and 7.15, respectively. The MALDI-TOF-TOF mass spectrum and N-terminal sequence 'DQHHSSKACC' suggested that the isolated RgPI is a member of the Bowman-Birk inhibitor family. RgPI exhibited noncompetitive type inhibitory activity against bovine pancreatic trypsin and chymotrypsin, with inhibition constants of 292 and 2265 nM, respectively. It was stable up to a temperature of 80 degrees C and was active over a wide pH range between 2 and 12. However, reduction with DTT or 2-mercaptoethanol resulted in loss of inhibitory activity against trypsin and chymotrypsin. It also decreased the activity of larval midgut trypsin-like proteinases in Manduca sexta . Its insecticidal property was further confirmed by reduction in the growth and development of these larvae, when supplemented in the diet.

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2

PubMed Central

Szláma, György; Trexler, Mária; Patthy, László

2013-01-01

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677–23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2. Structured digital abstract Furin cleaves Promyostatin by protease assay (View interaction) myostatin binds to PRO by surface plasmon resonance (View interaction) BMP-1 cleaves Promyostatin by protease assay (View interaction) ACR IIB physically interacts with Latent Myostatin by surface plasmon resonance (View interaction) Promyostatin and Promyostatin bind by comigration in gel electrophoresis (View interaction) WFIKKN1 binds to Latent Myostatin by pull down (View interaction) ACR IIB binds to Mature Myostatin by surface plasmon resonance (View Interaction: 1, 2, 3) WFIKKN1 binds to Myostatin Prodomain by surface plasmon resonance (View Interaction: 1, 2, 3) PMID:23829672

MicroRNA-96 Promotes Tumor Invasion in Colorectal Cancer via RECK.

PubMed

Iseki, Yasuhito; Shibutani, Masatsune; Maeda, Kiyoshi; Nagahara, Hisashi; Fukuoka, Tatsunari; Matsutani, Shinji; Hirakawa, Kosei; Ohira, Masaichi

2018-04-01

miR-96 is reported to inhibit reversion cysteine-rich Kazal motif (RECK), which is associated with tumor invasion, in solid cancer types (e.g. breast cancer, non-small cell lung cancer, esophageal cancer). The purpose of this study is to clarify whether miR-96 is similarly associated with tumor invasion in colorectal cancer. We performed western blotting to investigate the expression of RECK when miR-96 mimics or inhibitors were transferred into HCT-116 colorectal cancer cells. The RECK mRNA level was assessed by a reverse transcription polymerase chain reaction. An invasion assay was used to evaluate tumor invasion. The expression of RECK was inhibited by the transfection of miR-96 mimics. RECK mRNA level was reduced by miR-96 mimics and increased by miR-96 inhibitor. In the invasion assay, miR-96 mimics were shown to promote tumor invasion. miR-96 may be associated with tumor invasion through inhibition of RECK expression in colorectal cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Classical late infantile neuronal ceroid lipofuscinosis fibroblasts are deficient in lysosomal tripeptidyl peptidase I.

PubMed

Vines, D J; Warburton, M J

1999-01-25

Tripeptidyl peptidase I (TPP-I) is a lysosomal enzyme that cleaves tripeptides from the N-terminus of polypeptides. A comparison of TPP-I amino acid sequences with sequences derived from an EST database suggested that TPP-I is identical to a pepstatin-insensitive carboxyl proteinase of unknown specificity which is mutated in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disease. Both TPP-I and the carboxyl proteinase have an M(r) of about 46 kDa and are, or are predicted to be, resistant to inhibitors of the four major classes of proteinases. Fibroblasts from LINCL patients have less than 5% of the normal TPP-I activity. The activities of other lysosomal enzymes, including proteinases, are in the normal range. LINCL fibroblasts are also defective at degrading short polypeptides and this defect can be induced in normal fibroblasts by treatment with a specific inhibitor or TPP-I. These results suggest that the cell damage, especially neuronal, observed in LINCL results from the defective degradation and consequent lysosomal storage of small peptides.

Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches

NASA Astrophysics Data System (ADS)

Katritch, Vsevolod; Byrd, Chelsea M.; Tseitin, Vladimir; Dai, Dongcheng; Raush, Eugene; Totrov, Maxim; Abagyan, Ruben; Jordan, Robert; Hruby, Dennis E.

2007-10-01

Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays (˜20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K.

PubMed

Ali Mohammed, Marwan Mansoor; Nerland, Audun H; Al-Haroni, Mohammed; Bakken, Vidar

2013-01-01

Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM), often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I) and proteinase K. F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA) was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.

New insights on molecular interactions of organophosphorus pesticides with esterases.

PubMed

Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

2017-02-01

Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the organophosphorus toxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

PubMed

Yang, Tao-Tao; Cao, Na; Zhang, Hai-Hui; Wei, Jian-Bo; Song, Xiao-Xia; Yi, Dong-Min; Chao, Shuai-Heng; Zhang, Li-Da; Kong, Ling-Fei; Han, Shuang-Yin; Yang, Yu-Xiu; Ding, Song-Ze

2018-04-15

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased stepwise from nonatrophic gastritis to gastric cancer except slightly decrease in chronic atrophic gastritis group. In addition, we noted that histone H3 serine 10 phosphorylation staining is accompanied with its location changes from gastric gland bottom expanded to whole gland as disease stage progress. These results indicate that stepwise gastric carcinogenesis is associated with altered histone H3 serine 10 phosphorylation, Helicobacter pylori infection enhances histone H3 serine 10 phosphorylation expression in these processes; it is also accompanied with histone H3 serine 10 phosphorylation location change from gland bottom staining expand to whole gland expression. The results suggest that epigenetic dysregulation may play important roles in Helicobacter pylori-induced gastric cancer. © 2018 The Authors. Helicobacter Published by John Wiley & Sons Ltd.

[Phospholipase and proteinase production by Malassezia pachydermatis isolated in dogs with and without otitis].

PubMed

Ortiz, Gustavo; Martín, M Carmen; Carrillo-Muñoz, Alfonso J; Payá, M Jesús

2013-01-01

Malassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied. In the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis. The phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis. In our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%). Our results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis. Copyright © 2012 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Differential modulation of degradative and repair responses of interleukin-1-treated chondrocytes by platelet-derived growth factor.

PubMed Central

Harvey, A K; Stack, S T; Chandrasekhar, S

1993-01-01

Interleukin 1 (IL-1) plays a dual role in cartilage matrix degeneration by promoting extracellular proteinase action such as the matrix metalloproteinases (increased degradation) and by suppressing the synthesis of extracellular matrix molecules (inhibition of repair). Platelet-derived growth factor (PDGF) is a wound-healing hormone which is released along with IL-1 during the inflammatory response. Since previous studies have shown that PDGF enhances IL-1 alpha effects on metalloproteinase activity, in this report, we have examined whether PDGF modifies IL-1 beta effects on cartilage proteoglycan synthesis. Initially, we confirmed that rabbit articular chondrocytes treated with IL-1 beta + PDGF induced higher proteinase activity, in comparison with IL-1-treated cells. We further observed that the increased proteinase activity correlated with an increase in the synthesis of collagenase/stromelysin proteins and a corresponding increase in the steady-state mRNA levels for both the enzymes. Studies on IL-1 receptor expression suggested that PDGF caused an increase in IL-1 receptor expression which, by augmenting the IL-1 response, may have led to the increase in proteinase induction. Analysis of proteoglycan synthesis confirmed that IL-1 reduced the incorporation of sulphated proteoglycan, aggrecan, into the extracellular matrix of chondrocytes, whereas PDGF stimulated it. However, cells treated with IL-1 + PDGF synthesized normal levels of aggrecan. This is in contrast with cells treated with IL-1 + fibroblast growth factor, in which case only proteinase activity was potentiated. The results allow us to conclude that (a) the two effector functions that play a role in matrix remodelling, namely matrix lysis (proteinase induction) and matrix repair (proteoglycan synthesis), occur via distinct pathways and (b) PDGF may play a crucial role in cartilage repair by initially causing matrix degradation followed by promoting new matrix synthesis. Images Figure 1 Figure 2 Figure 5 Figure 6 PMID:8503839

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

PubMed

Jagdeo, Julienne M; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M; Jan, Eric

2018-04-15

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed t erminal a mine i sotopic l abeling of s ubstrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3C pro s) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3C pro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3C pro -targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3C pro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3C pro substrates in vivo , we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection. Copyright © 2018 Jagdeo et al.

Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp (Ctenopharyngodon idellus).

PubMed

Wu, Jiu-Lin; Ge, Shang-Ying; Cai, Zhi-Xing; Liu, Hui; Liu, Ying-Xiang; Wang, Jian-Hua; Zhang, Qi-Qing

2014-02-15

A gelatinolytic matrix metalloproteinase (gMMP) from grass carp skeletal muscle was purified by 30-70% ammonium sulphate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, and affinity on gelatin-sepharose. The molecular weight of the proteinase as estimated by SDS-PAGE was 70 kDa under non-reducing conditions. The enzyme revealed high activity from 30 to 50 °C, and the gelatin hydrolysing activity was investigated at a slightly alkaline pH range using gelatin as substrate. Metalloproteinase inhibitor EDTA completely suppressed the gelatinolytic activity, while other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca(2+) was essential for the gelatinolytic activity. Further, peptide mass fingerprinting obtained four fragments with 45 amino acid residues, which were highly identical to MMP-2 from fish species. The gMMP could effectively hydrolyse type I collagen even at 4 °C, suggesting its involvement in the texture softening of fish muscle during the post-mortem stage. Copyright © 2013 Elsevier Ltd. All rights reserved.

The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity.

PubMed

Kroczynska, Barbara; Evangelista, Christina M; Samant, Shalaka S; Elguindi, Ebrahim C; Blond, Sylvie Y

2004-03-19

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.

Zoanthid mucus as new source of useful biologically active proteins.

PubMed

Guarnieri, Míriam Camargo; de Albuquerque Modesto, Jeanne Claíne; Pérez, Carlos Daniel; Ottaiano, Tatiana Fontes; Ferreira, Rodrigo da Silva; Batista, Fabrício Pereira; de Brito, Marlon Vilela; Campos, Ikaro Henrique Mendes Pinto; Oliva, Maria Luiza Vilela

2018-03-01

Palythoa caribaeorum is a very common colonial zoanthid in the coastal reefs of Brazil. It is known for its massive production of mucus, which is traditionally used in folk medicine by fishermen in northeastern Brazil. This study identified biologically active compounds in P. caribaerum mucus. Crude mucus was collected during low tides by the manual scraping of colonies; samples were maintained in an ice bath, homogenized, and centrifuged at 16,000 g for 1 h at 4 °C; the supernatant (mucus) was kept at -80 °C until use. The enzymatic (proteolytic and phospholipase A 2 ), inhibitory (metallo, cysteine and serine proteases), and hemagglutinating (human erythrocyte) activities were determined. The results showed high levels of cysteine and metallo proteases, intermediate levels of phosholipase A 2 , low levels of trypsin, and no elastase and chymotrypsin like activities. The mucus showed potent inhibitory activity on snake venom metalloproteases and cysteine proteinase papain. In addition, it showed agglutinating activity towards O + , B + , and A + erythrocyte types. The hemostatic results showed that the mucus prolongs the aPTT and PT, and strongly inhibited platelet aggregation induced by arachidonic acid, collagen, epinephrine, ADP, and thrombin. The antimicrobial activity was tested on 15 strains of bacteria and fungi through the radial diffusion assay in agar, and no activity was observed. Compounds in P. caribaeorum mucus were analyzed for the first time in this study, and our results show potential pharmacological activities in these compounds, which are relevant for use in physiopathological investigations. However, the demonstration of these activities indicates caution in the use of crude mucus in folk medicine. Furthermore, the present or absent activities identified in this mucus suggest that the studied P. caribaeorum colonies were in thermal stress conditions at the time of sample collection; these conditions may precede the bleaching process in zoanthids. Hence, the use of mucus as an indicator of this process should be evaluated in the future. Copyright © 2018. Published by Elsevier Ltd.

The solvation of L-serine in mixtures of water with some aprotic solvents at 298.15 K

NASA Astrophysics Data System (ADS)

Mezhevoi, I. N.; Badelin, V. G.

2009-03-01

The integral enthalpies of solution Δsol H m of L-serine in mixtures of water with acetonitrile, 1,4-dioxane, dimethylsulfoxide (DMSO), and acetone were measured by solution calorimetry at organic component concentrations up to 0.31 mole fractions. The standard enthalpies of solution (Δsol H°), transfer (Δtr H°), and solvation (Δsolv H°) of L-serine from water into mixed solvents were calculated. The dependences of Δsol H°, Δsolv H°, and Δtr H° on the composition of aqueous-organic solvents contained extrema. The calculated enthalpy coefficients of pair interactions of the amino acid with cosolvent molecules were positive and increased in the series acetonitrile, 1,4-dioxane, DMSO, acetone. The results obtained were interpreted from the point of view of various types of interactions in solutions and the influence of the nature of organic solvents on the thermochemical characteristics of solutions.

Epithelial Integrity Is Maintained by a Matriptase-Dependent Proteolytic Pathway

PubMed Central

List, Karin; Kosa, Peter; Szabo, Roman; Bey, Alexandra L.; Wang, Chao Becky; Molinolo, Alfredo; Bugge, Thomas H.

2009-01-01

A pericellular proteolytic pathway initiated by the transmembrane serine protease matriptase plays a critical role in the terminal differentiation of epidermal tissues. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of this membrane serine protease in general epithelial homeostasis. Here we generated mice with conditional deletion of the St14 gene, encoding matriptase, and show that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. The study reveals that the homeostasis of multiple simple and stratified epithelia is matriptase-dependent, and provides an important animal model for the exploration of this membrane serine protease in a range of physiological and pathological processes. PMID:19717635

Regulation of Hemolysin Expression and Virulence of Staphylococcus aureus by a Serine/Threonine Kinase and Phosphatase

PubMed Central

Burnside, Kellie; Lembo, Annalisa; de los Reyes, Melissa; Iliuk, Anton; BinhTran, Nguyen-Thao; Connelly, James E.; Lin, Wan-Jung; Schmidt, Byron Z.; Richardson, Anthony R.; Fang, Ferric C.; Tao, Weiguo Andy; Rajagopal, Lakshmi

2010-01-01

Exotoxins, including the hemolysins known as the alpha (α) and beta (β) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding α toxin was decreased in a Δstp1 mutant strain and increased in a Δstk1 strain. Microarray analysis of a Δstk1 mutant revealed increased transcription of additional exotoxins. A Δstp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Δstk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Δstk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence. PMID:20552019

Alveolar Macrophages Play a Key Role in Cockroach-Induced Allergic Inflammation via TNF-α Pathway

PubMed Central

Kim, Joo Young; Sohn, Jung Ho; Choi, Je-Min; Lee, Jae-Hyun; Hong, Chein-Soo; Lee, Joo-Shil; Park, Jung-Won

2012-01-01

The activity of the serine protease in the German cockroach allergen is important to the development of allergic disease. The protease-activated receptor (PAR)-2, which is expressed in numerous cell types in lung tissue, is known to mediate the cellular events caused by inhaled serine protease. Alveolar macrophages express PAR-2 and produce considerable amounts of tumor necrosis factor (TNF)-α. We determined whether the serine protease in German cockroach extract (GCE) enhances TNF-α production by alveolar macrophages through the PAR-2 pathway and whether the TNF-α production affects GCE-induced pulmonary inflammation. Effects of GCE on alveolar macrophages and TNF-α production were evaluated using in vitro MH-S and RAW264.6 cells and in vivo GCE-induced asthma models of BALB/c mice. GCE contained a large amount of serine protease. In the MH-S and RAW264.7 cells, GCE activated PAR-2 and thereby produced TNF-α. In the GCE-induced asthma model, intranasal administration of GCE increased airway hyperresponsiveness (AHR), inflammatory cell infiltration, productions of serum immunoglobulin E, interleukin (IL)-5, IL-13 and TNF-α production in alveolar macrophages. Blockade of serine proteases prevented the development of GCE induced allergic pathologies. TNF-α blockade also prevented the development of such asthma-like lesions. Depletion of alveolar macrophages reduced AHR and intracellular TNF-α level in pulmonary cell populations in the GCE-induced asthma model. These results suggest that serine protease from GCE affects asthma through an alveolar macrophage and TNF-α dependent manner, reflecting the close relation of innate and adaptive immune response in allergic asthma model. PMID:23094102

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates

PubMed Central

Koga, Reiko; Miyoshi, Yurika; Sakaue, Hiroaki; Hamase, Kenji; Konno, Ryuichi

2017-01-01

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties. PMID:29255714

DOE Office of Scientific and Technical Information (OSTI.GOV)

E Nistal-Villan; M Gack; G Martinez-Delgado

RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity hasmore » not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.« less

Effects of cysteine proteinase inhibitors scN and E-64 on southern corn rootworm larval development

USDA-ARS?s Scientific Manuscript database

The southern corn rootworm (SCRW) can be a serious pest of peanut pods. A laboratory bioassay was developed to test feeding cysteine proteinase inhibitors soyacystatin N (scN) and E-64 against southern corn rootworm reared on artificial diet to determine the effects on larvae development and mortal...

[Experimental approach to the prophylaxis and treatment of acute lung injury syndrome with proteinase inhibitors and corvitin].

PubMed

Moĭbenko, O O; Kubyshkin, A V; Kharchenko, V Z; Horokhova, N Iu; Semenets', P F

2003-01-01

The results of a combined study of the proteolysis on a model of post-ischemic toxemia in rats showed a decrease in antiproteinase potential and an activation of proteolysis. The activation of proteolysis and inhibition of antiproteinases was observed not only in the blood, but also in the bronchoalveolar secretion. Those changes were accompanied with the changes in the morphological structure of the lungs. The data obtained have shown a high effectiveness of proteinase inhibitor (contrical) and an antioxidant of flavonoid group (corvetine). Those drugs decreased the morphological changes in the lungs and prevented the development of imbalance in proteinase-inhibitor system. The prophylactic effect was more considerable when both drugs were used in a combined way.

Extracellular metalloproteinases in Phytomonas serpens.

PubMed

Vermelho, Alane B; Almeida, Flávia V S; Bronzato, Leandro S; Branquinha, Marta H

2003-03-01

The detection of extracellular proteinases in Phytomonas serpens, a trypanosomatid isolated from tomato fruits, is demonstrated in this paper. Maximal production occurred at the end of the logarithmic phase of growth. These enzymes exhibited selective substrate utilization in SDS-PAGE, being more active with gelatin; hemoglobin and bovine serum albumin were not degraded. Three proteinases were detected in SDS-PAGE-gelatin, with apparent molecular masses between 94 and 70 kDa. The proteolytic activity was completely blocked by 1,10-phenanthroline and strongly inhibited by EDTA, whereas a partial inhibition was observed with trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) and soybean trypsin inhibitor; phenylmethylsulfonyl fluoride weakly inhibited the enzymes. This inhibition profile indicated that these extracellular proteinases belong to the metalloproteinase class.

Brewer’s spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

PubMed Central

do Nascimento, Rodrigo Pires; Junior, Nelson Alves; Coelho, Rosalie Reed Rodrigues

2011-01-01

Brewer’s spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates. PMID:24031767

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

[Enzymatic activity, slime production and antifungal agent sensitivity of Candida sp].

PubMed

Silva, Jaqueline Otero; Ferreira, Joseane Cristina; Candido, Regina Célia

2007-01-01

Abilith of Candida spp to secrete extracellular enzymes and slime has been associated as pathogenicity factors. Out of a total of 37 strains of Candida sp, 100% were proteinase producers, 83.8% were phospholipase producers, 64.9% were slime producers and 100% were sensitive to fluconazole and itraconazole. Seventeen typings (enzymes/slime) were found. This methodology presented a good discrimination rate (D=0.93) and could be used for phenotypic characterization of yeasts.

Serine biosynthesis and transport defects.

PubMed

El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of a type II cystatin in Fragaria chiloensis: A proteinase inhibitor differentially regulated during achene development and in response to biotic stress-related stimuli.

PubMed

Aceituno-Valenzuela, Uri; Covarrubias, María Paz; Aguayo, María Francisca; Valenzuela-Riffo, Felipe; Espinoza, Analía; Gaete-Eastman, Carlos; Herrera, Raúl; Handford, Michael; Norambuena, Lorena

2018-05-19

The equilibrium between protein synthesis and degradation is key to maintaining efficiency in different physiological processes. The proteinase inhibitor cystatin regulates protease activities in different developmental and physiological contexts. Here we describe for the first time the identification and the biological function of the cysteine protease inhibitor cystatin of Fragaria chiloensis, FchCYS1. Based on primary sequence and 3D-structural homology modelling, FchCYS1 is a type II phytocystatin with high identity to other cystatins of the Fragaria genus. Both the papain-like and the legumain-like protease inhibitory domains are indeed functional, based on in vitro assays performed with Escherichia coli protein extracts containing recombinant FchCYS1. FchCYS1 is differentially-expressed in achenes of F. chiloensis fruits, with highest expression as the fruit reaches the ripened stage, suggesting a role in preventing degradation of storage proteins that will nourish the embryo during seed germination. Furthermore, FchCYS1 responds transcriptionally to the application of salicylic acid and to mechanical injury, strongly suggesting that FchCYS1 could be involved in the response against pathogen attack. Overall these results point to a role for FchCYS1 in diverse physiological processes in F. chiloensis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster

PubMed Central

Nagasawa, Tatsuki; Sano, Kaori; Kawaguchi, Mari; Kobayashi, Ken-ichiro; Yasumasu, Shigeki; Inokuchi, Tomofumi

2016-01-01

Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37–38 kDa and 31–34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians. PMID:26711235

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 inmore » 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. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.« less

Novel Computational Protocols for Functionally Classifying and Characterising Serine Beta-Lactamases

PubMed Central

Das, Sayoni; Dawson, Natalie L.; Dobrijevic, Dragana; Orengo, Christine

2016-01-01

Beta-lactamases represent the main bacterial mechanism of resistance to beta-lactam antibiotics and are a significant challenge to modern medicine. We have developed an automated classification and analysis protocol that exploits structure- and sequence-based approaches and which allows us to propose a grouping of serine beta-lactamases that more consistently captures and rationalizes the existing three classification schemes: Classes, (A, C and D, which vary in their implementation of the mechanism of action); Types (that largely reflect evolutionary distance measured by sequence similarity); and Variant groups (which largely correspond with the Bush-Jacoby clinical groups). Our analysis platform exploits a suite of in-house and public tools to identify Functional Determinants (FDs), i.e. residue sites, responsible for conferring different phenotypes between different classes, different types and different variants. We focused on Class A beta-lactamases, the most highly populated and clinically relevant class, to identify FDs implicated in the distinct phenotypes associated with different Class A Types and Variants. We show that our FunFHMMer method can separate the known beta-lactamase classes and identify those positions likely to be responsible for the different implementations of the mechanism of action in these enzymes. Two novel algorithms, ASSP and SSPA, allow detection of FD sites likely to contribute to the broadening of the substrate profiles. Using our approaches, we recognise 151 Class A types in UniProt. Finally, we used our beta-lactamase FunFams and ASSP profiles to detect 4 novel Class A types in microbiome samples. Our platforms have been validated by literature studies, in silico analysis and some targeted experimental verification. Although developed for the serine beta-lactamases they could be used to classify and analyse any diverse protein superfamily where sub-families have diverged over both long and short evolutionary timescales. PMID:27332861

Snake venomics of the pit vipers Porthidium nasutum, Porthidium ophryomegas, and Cerrophidion godmani from Costa Rica: toxicological and taxonomical insights.

PubMed

Lomonte, Bruno; Rey-Suárez, Paola; Tsai, Wan-Chih; Angulo, Yamileth; Sasa, Mahmood; Gutiérrez, José María; Calvete, Juan J

2012-02-16

Within the Neotropical pit vipers, a lineage of primarily Middle American snake species referred to as the "Porthidium group" includes the genera Atropoides, Cerrophidion, and Porthidium. In this study, the venom proteomes of Porthidium nasutum, P. ophryomegas, and Cerrophidion godmani from Costa Rica were analyzed, and correlated to their toxic and enzymatic activities. Their HPLC profiles revealed a higher similarity between the two Porthidium species than between these and C. godmani. Proteins belonging to nine (P. nasutum), eight (P. ophryomegas), and nine (C. godmani) families were identified by mass spectrometry or N-terminal sequencing. Final cataloging of proteins and their relative abundances confirmed the close relationship between venoms of P. nasutum and P. ophryomegas, departing from that of C. godmani. Since the latter species had been taxonomically classified as Porthidium godmani previously, our venomic analyses agree with its current generic status. Venoms of P. nasutum and P. ophryomegas, despite containing abundant metalloproteinases and serine proteinases, lack procoagulant activity on human plasma, in contrast to venom of C. godmani. The latter induced strong myotoxicity in mice, which correlates with its high proportion of phospholipases A(2), whereas venoms from the two Porthidium species, containing lower amounts of these enzymes, induced only mild muscle damage. Copyright © 2011 Elsevier B.V. All rights reserved.

Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme from Gloydius saxatilis venom

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Wenqing; Zhao, Wei; Key Laboratory of Structural Biology, Chinese Academy of Sciences, 96 Jinzhai Road, Hefei, Anhui 230027

2007-08-01

The thrombin-like enzyme saxthrombin has been purified from G. saxatilis snake venom. Crystallization conditions were found and a data set was obtained to 1.43 Å. The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatographymore » procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS–PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 Å. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 Å, β = 96.72°, and the Matthews coefficient (V{sub M}) was calculated to be 2.13 Å{sup 3} Da{sup −1} with one molecule in the asymmetric unit.« less

In Vivo d-Serine Hetero-Exchange through Alanine-Serine-Cysteine (ASC) Transporters Detected by Microelectrode Biosensors

PubMed Central

2013-01-01

d-Serine, a co-agonist of N-methyl d-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. d-Serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular d-serine levels in vivo are still unclear. d-Serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of d-serine extracellular concentration in vivo. Alternatively, exocytotic d-serine release has also been proposed. In this study, the dynamics of d-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of d-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. d-Serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in d-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release d-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased d-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that d-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of d-serine extracellular concentration. PMID:23581544

Modulation of serpin reaction through stabilization of transient intermediate by ligands bound to alpha-helix F.

PubMed

Komissarov, Andrey A; Zhou, Aiwu; Declerck, Paul J

2007-09-07

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways.

Mutation at p53 serine 389 does not rescue the embryonic lethality in mdm2 or mdm4 null mice.

PubMed

Iwakuma, Tomoo; Parant, John M; Fasulo, Mark; Zwart, Edwin; Jacks, Tyler; de Vries, Annemieke; Lozano, Guillermina

2004-10-07

Mdm2 and its homolog Mdm4 inhibit the function of the tumor suppressor p53. Targeted disruption of either mdm2 or mdm4 genes in mice results in embryonic lethality that is completely rescued by concomitant deletion of p53, suggesting that deletion of negative regulators of p53 results in a constitutively active p53. Thus, these mouse models offer a unique in vivo system to assay the functional significance of different p53 modifications. Phosphorylation of serine 389 in murine p53 occurs specifically after ultraviolet-light-induced DNA damage, and phosphorylation of this site enhances p53 activity both in vitro and in vivo. Recently, mice with a serine to alanine substitution at serine 389 (p53S389A) in the endogenous p53 locus were generated. To examine the in vivo significance of serine 389 phosphorylation during embryogenesis, we crossed these mutant mice to mice lacking mdm2 or mdm4. The p53S389A allele did not alter the embryonic lethality of mdm2 or mdm4. Additional crosses to assay the effect of one p53S389A allele with a p53 null allele also did not rescue the lethal phenotypes. In conclusion, the phenotypes due to loss of mdm2 or mdm4 were not even partially rescued by p53S389A, suggesting that p53S389A is functionally wild type during embryogenesis.

Role of serine threonine protein phosphatase type 5 (PP5) in the regulation of stress induced signaling networks and cancer

PubMed Central

Golden, Teresa; Swingle, Mark; Honkanen, Richard E.

2008-01-01

Although the aberrant actions of protein kinases have long been known to contribute to tumor promotion and carcinogenesis, roles for proteins phosphatases in the development of human cancer have only emerged in the last decade. In this review, we discuss the data obtained from studies examining the biological and pathological roles of a serine/threonine protein phosphate, PP5, which suggest that PP5 is a potentially important regulator of both hormone- and stress-induced networks that enable a cell to respond appropriately to genomic stress. PMID:18253812

Metabolomic Analysis of the Secretome of Human Embryonic Stem Cells Following Methyl Parathion and Methyl Paraoxon Exposure, Phase 1: Initial Nontargeted LC-MS

DTIC Science & Technology

2014-01-01

and threonine metabolism 6 40 0.006188 0.10 MP H hsa00260 Glycine, serine and threonine metabolism 3 40 0.010283 0.07 MP H hsa00280 Valine, leucine...hsa00770 Pantothenate and CoA biosynthesis 5 24 0.005066 0.07 MP M hsa04142 Lysosome 2 4 0.002426 0.10 MPO M hsa04930 Type II diabetes mellitus 3 5...of MPO and increased after exposure to all three dose levels of MP. The glycine, serine and threonine metabolism pathway is of interest because

β-Amino acid catalyzed asymmetric Michael additions: design of organocatalysts with catalytic acid/base dyad inspired by serine proteases.

PubMed

Yang, Hui; Wong, Ming Wah

2011-09-16

A new type of chiral β-amino acid catalyst has been computationally designed, mimicking the enzyme catalysis of serine proteases. Our catalyst approach is based on the bioinspired catalytic acid/base dyad, namely, a carboxyl and imidazole pair. DFT calculations predict that this designed organocatalyst catalyzes Michael additions of aldehydes to nitroalkenes with excellent enantioselectivities and remarkably high anti diastereoselectivities. The unusual stacked geometry of the enamine intermediate, hydrogen bonding network, and the adoption of an exo transition state are the keys to understand the stereoselectivity. © 2011 American Chemical Society

Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

PubMed Central

Bartlett, John D.

2013-01-01

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

Thick-tissue bioreactor as a platform for long-term organotypic culture and drug delivery.

PubMed

Markov, Dmitry A; Lu, Jenny Q; Samson, Philip C; Wikswo, John P; McCawley, Lisa J

2012-11-07

We have developed a novel, portable, gravity-fed, microfluidics-based platform suitable for optical interrogation of long-term organotypic cell culture. This system is designed to provide convenient control of cell maintenance, nutrients, and experimental reagent delivery to tissue-like cell densities housed in a transparent, low-volume microenvironment. To demonstrate the ability of our Thick-Tissue Bioreactor (TTB) to provide stable, long-term maintenance of high-density cellular arrays, we observed the morphogenic growth of human mammary epithelial cell lines, MCF-10A and their invasive variants, cultured under three-dimensional (3D) conditions inside our system. Over the course of 21 days, these cells typically develop into hollow "mammospheres" if cultured in standard 3D Matrigel. This complex morphogenic process requires alterations in a variety of cellular functions, including degradation of extracellular matrix that is regulated by cell-produced matrix proteinases. For our "drug" delivery testing and validation experiments we have introduced proteinase inhibitors into the fluid supply system, and we observed both reduced proteinase activity and inhibited cellular morphogenesis. The size inhibition results correlated well with the overall proteinase activities of the tested cells.

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

PubMed

Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

2012-02-10

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. Copyright © 2011 Elsevier B.V. All rights reserved.

Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes, Micrurus altirostris and M. corallinus.

PubMed

Corrêa-Netto, Carlos; Junqueira-de-Azevedo, Inácio de L M; Silva, Débora A; Ho, Paulo L; Leitão-de-Araújo, Moema; Alves, Maria Lúcia M; Sanz, Libia; Foguel, Débora; Zingali, Russolina Benedeta; Calvete, Juan J

2011-08-24

The venom proteomes of Micrurus altirostris and M. corallinus were analyzed by combining snake venomics and venom gland transcriptomic surveys. In both coral snake species, 3FTx and PLA(2) were the most abundant and diversified toxin families. 33 different 3FTxs and 13 PLA(2) proteins, accounting respectively for 79.5% and 13.7% of the total proteins, were identified in the venom of M. altirostris. The venom of M. corallinus comprised 10 3FTx (81.7% of the venom proteome) and 4 (11.9%) PLA(2) molecules. Transcriptomic data provided the full-length amino acid sequences of 18 (M. altirostris) and 10 (M. corallinus) 3FTxs, and 3 (M. altirostris) and 1 (M. corallinus) novel PLA(2) sequences. In addition, venom from each species contained single members of minor toxin families: 3 common (PIII-SVMP, C-type lectin-like, L-amino acid oxidase) and 4 species-specific (CRISP, Kunitz-type inhibitor, lysosomal acid lipase in M. altirostris; serine proteinase in M. corallinus) toxin classes. The finding of a lipase (LIPA) in the venom proteome and in the venom gland transcriptome of M. altirostris supports the view of a recruitment event predating the divergence of Elapidae and Viperidae more than 60 Mya. The toxin profile of both M. altirostris and M. corallinus venoms points to 3FTxs and PLA(2) molecules as the major players of the envenoming process. In M. altirostris venom, all major, and most minor, 3FTxs display highest similarity to type I α-neurotoxins, suggesting that these postsynaptically acting toxins may play the predominant role in the neurotoxic effect leading to peripheral paralysis, respiratory arrest, and death. M. corallinus venom posesses both, type I α-neurotoxins and a high-abundance (26% of the venom proteome) protein of subfamily XIX of 3FTxs, exhibiting similarity to bucandin from Malayan krait, Bungarus candidus, venom, which enhances acetylcholine release presynaptically. This finding may explain the presynaptic neurotoxicity of M. corallinus venom and the lack of this effect in M. altirostris venom. The anti-Micrurus (corallinus and frontalis) antivenom produced by Instituto Butantan quantitatively immunodepleted the minor toxins from M. altirostris and M. corallinus venoms but showed impaired crossreactivity towards their major 3FTx and PLA(2) molecules. The structural diversity of 3FTxs among Micrurus sp. may underlay the impaired cross-immunoreactivity of the Butantan antivenom towards M. altirostris and M. corallinus toxins, hampering the possibility to raise an antivenom against a simple venom mixture exhibiting paraspecific neutralization of other Micrurus venoms. Copyright © 2011 Elsevier B.V. All rights reserved.

Construction of Escherichia coli strains producing L-serine from glucose.

PubMed

Li, Yu; Chen, Gu-Kui; Tong, Xin-Wei; Zhang, Hui-Tu; Liu, Xiao-Guang; Liu, Yi-Han; Lu, Fu-Ping

2012-08-01

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

PubMed

Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

2014-01-01

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and Secreted Aspartyl Proteinase (SAPS) 2, 4, 5 and 6 Expressions

DTIC Science & Technology

2015-04-01

AWARD NUMBER: W81XWH-12-2-0025 TITLE: Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue and C. albicans Growth, Transition and...REPORT TYPE Annual 3. DATES COVERED 1 Apr 2014 - 31 Mar 2015 4. TITLE AND SUBTITLE Effect of Antimicrobial Peptide KSL-W on Human Gingival Tissue...of new antifungal drugs using various synthetic and naturally occurring antimicrobial molecules. Natural antimicrobial peptides , such as defensins

Smallpox Antiviral Drug

DTIC Science & Technology

2006-01-01

preparing a Continuation in Part ( CIP ) to add the new I7L cleavage assays recently developed by SIGA. Conclusions By using homology-based... developmental cycle . RNA viruses and retroviruses commonly undergo formative proteolysis in which large polyproteins are cleaved by viral encoded proteinases to...structural model of the vaccinia virus (VV) I7L proteinase was developed at Transtech Pharma. A unique chemical library of ~ 51,000 compounds was

D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability

PubMed Central

Lin, Hong; Jacobi, Ariel A.; Anderson, Stewart A.; Lynch, David R.

2016-01-01

D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating that D-serine effects are mediated through postsynaptic NMDARs. Conversely, exogenous application of glycine has no such effects, suggesting D-serine, rather than glycine, modulates postsynaptic events. Taken together, our findings demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development, implicating D-serine/SR as regulators of cortical synaptic and circuit development. PMID:26941605

D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability.

PubMed

Lin, Hong; Jacobi, Ariel A; Anderson, Stewart A; Lynch, David R

2016-01-01

D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating that D-serine effects are mediated through postsynaptic NMDARs. Conversely, exogenous application of glycine has no such effects, suggesting D-serine, rather than glycine, modulates postsynaptic events. Taken together, our findings demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development, implicating D-serine/SR as regulators of cortical synaptic and circuit development.

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

PubMed Central

Koutsoudakis, George; Urbanowicz, Richard A.; Mirza, Deeman; Ginkel, Corinne; Riebesehl, Nina; Calland, Noémie; Albecka, Anna; Price, Louisa; Hudson, Natalia; Descamps, Véronique; Backx, Matthijs; McClure, C. Patrick; Duverlie, Gilles; Pecheur, Eve-Isabelle; Dubuisson, Jean; Perez-del-Pulgar, Sofia; Forns, Xavier; Steinmann, Eike; Tarr, Alexander W.; Pietschmann, Thomas

2014-01-01

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codon-switching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic “fossil record” of historical purifying selection against E1E2 intermediate phenotypes. PMID:24173227

Reduction of infection by inhibiting mTOR pathway is associated with reversing repression of type I IFN by PRRSV

USDA-ARS?s Scientific Manuscript database

Type I interferons (IFNs) are critical in animal antiviral regulation. IFN-mediated signaling regulates hundreds of genes that are directly associated with antiviral, immune and other physiological responses. The signaling pathway mediated by mechanistic target of rapamycin (mTOR), a serine/threonin...

Exoenzyme activity and possibility identification of Candida dubliniensis among Candida albicans species isolated from vaginal candidiasis.

PubMed

Jafari, Maryam; Salari, Samira; Pakshir, Keyvan; Zomorodian, Kamiar

2017-09-01

Vulvovaginal candidiasis (VVC) or vaginal candidiasis is a common fungal infection of the genitals causing inflammation, irritation, itching, and vaginal discharge. Common yeast infections are caused by the yeast species C. albicans. However, there are other species of Candida such as C. dubliniensis which are considered as the causative agents of this infection. Hydrolytic enzymes such as proteinase and coagulase are known as virulence factors. The aim of this study was the molecular confirmation and differentiation of C. dubliniensis among C. albicans strains isolated from women with vulvovaginal candidiasis by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) and the evaluation of proteinase and coagulase activities. A total of 100 C. albicans strains isolated from women with vulvovaginal candidiasis referred to Shiraz medical clinics were enrolled in the study. All the isolates were primarily identified by conventional methods. PCR-RFLP method was used for the confirmation and identification of C. albicans and C. dubliniensis. Moreover, in vitro proteinase and coagulase activities of these isolates were evaluated using bovine serum albumin media and classical rabbit plasma tube test. As a result, PCR-RFLP identified 100% of the isolates as C. albicans, and no C. dubliniensis could be identified in this study. 84% of the isolates showed proteinase activity, whereas coagulase activity was only detected in 5% of the isolates. This study reveals that C. dubliniensis plays no role in vaginal candidiasis in Iranian patients. Proteinase production could be an essential virulence factor in C. albicans pathogenicity, but coagulase activity has less potential in this matter. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetically Distinct Subsets within ANCA-Associated Vasculitis

PubMed Central

Lyons, Paul A.; Rayner, Tim F.; Trivedi, Sapna; Holle, Julia U.; Watts, Richard A.; Jayne, David R.W.; Baslund, Bo; Brenchley, Paul; Bruchfeld, Annette; Chaudhry, Afzal N.; Tervaert, Jan Willem Cohen; Deloukas, Panos; Feighery, Conleth; Gross, Wolfgang L.; Guillevin, Loic; Gunnarsson, Iva; P, Lorraine Harper M.R.C; Hrušková, Zdenka; Little, Mark A.; Martorana, Davide; Neumann, Thomas; Ohlsson, Sophie; Padmanabhan, Sandosh; Pusey, Charles D.; Salama, Alan D.; Sanders, Jan-Stephan F.; Savage, Caroline O.; Segelmark, Mårten; Stegeman, Coen A.; Tesař, Vladimir; Vaglio, Augusto; Wieczorek, Stefan; Wilde, Benjamin; Zwerina, Jochen; Rees, Andrew J.; Clayton, David G.; Smith, Kenneth G.C.

2013-01-01

BACKGROUND Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis. METHODS A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria. RESULTS We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti–proteinase 3 ANCA was associated with HLA-DP and the genes encoding α1-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P = 6.2×10−89, P = 5.6×10−12, and P = 2.6×10−7, respectively). Anti–myeloperoxidase ANCA was associated with HLA-DQ (P = 2.1×10−8). CONCLUSIONS This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA–associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA–associated vasculitis and myeloperoxidase ANCA–associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.) PMID:22808956

A supramolecular complex between proteinases and beta-cyclodextrin that preserves enzymatic activity: physicochemical characterization.

PubMed

Denadai, Angelo M L; Santoro, Marcelo M; Lopes, Miriam T P; Chenna, Angélica; de Sousa, Frederico B; Avelar, Gabriela M; Gomes, Marco R Túlio; Guzman, Fanny; Salas, Carlos E; Sinisterra, Rubén D

2006-01-01

Cyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and wound-healing properties. In this study, we analyzed the association between beta-cyclodextrin (betaCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity. The physicochemical analyses suggest the formation of a complex between P1G10 and betaCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10 : betaCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and betaCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar betaCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29. The reported results underscore the potential for betaCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.

Molecular and biochemical characterisation of two aspartic proteinases TcAP1 and TcAP2 from Theobroma cacao seeds.

PubMed

Laloi, Maryse; McCarthy, James; Morandi, Olivia; Gysler, Christof; Bucheli, Peter

2002-09-01

Aspartic proteinase (EC 3.4.23) activity plays a pivotal role in the degradation of Theobroma cacao L. seed proteins during the fermentation step of cacao bean processing. Therefore, this enzyme is believed to be critical for the formation of the peptide and amino acid cocoa flavor precursors that occurs during fermentation. Using cDNA cloning and northern blot analysis, we show here that there are at least two distinct aspartic proteinase genes ( TcAP1 and TcAP2) expressed during cacao seed development. Both genes are expressed early during seed development and their mRNA levels decrease towards the end of seed maturation. TcAP2 is expressed at a much higher level than TcAP1, although the expression of TcAP1 increases slightly during germination. The proteins encoded by TcAP1 and TcAP2 are relatively different from each other (73% identity). This, and the fact that the two corresponding genes have different expression patterns, suggests that the TcAP1 and TcAP2 proteins may have different functions in the maturing seeds and during germination. Because the TcAP2 gene is expressed at a much higher level during seed development than TcAP1, it is likely that the TcAP2 protein is primarily responsible for the majority of the industrially important protein hydrolysis that occurs during cacao bean fermentation. Finally, TcAP2 has been functionally expressed in the yeast Yarrowia lipolytica. The secreted recombinant protein is able to hydrolyse bovine haemoglobin at acidic pH and is sensitive to pepstatin A, confirming that TcAP2 encodes an aspartic proteinase, and strongly suggests that this gene encodes the well-characterized aspartic proteinase of mature cacao seeds.

The anthelmintic efficacy of plant-derived cysteine proteinases against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, in vivo.

PubMed

Stepek, G; Lowe, A E; Buttle, D J; Duce, I R; Behnke, J M

2007-09-01

Gastrointestinal (GI) nematodes are important disease-causing organisms, controlled primarily through treatment with synthetic drugs, but the efficacy of these drugs has declined due to widespread resistance, and hence new drugs, with different modes of action, are required. Some medicinal plants, used traditionally for the treatment of worm infections, contain cysteine proteinases known to damage worms irreversibly in vitro. Here we (i) confirm that papaya latex has marked efficacy in vivo against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, (ii) demonstrate the dose-dependent nature of the activity (>90% reduction in egg output and 80% reduction in worm burden at the highest active enzyme concentration of 133 nmol), (iii) establish unequivocally that it is the cysteine proteinases that are the active principles in vivo (complete inhibition of enzyme activity when pre-incubated with the cysteine proteinase-specific inhibitor, E-64) and (iv) show that activity is confined to worms that are in the intestinal lumen. The mechanism of action was distinct from all current synthetic anthelmintics, and was the same as that in vitro, with the enzymes attacking and digesting the protective cuticle. Treatment had no detectable side-effects on immune cell numbers in the mucosa (there was no difference in the numbers of mast cells and goblet cells between the treated groups) and mucosal architecture (length of intestinal villi). Only the infected and untreated mice had much shorter villi than the other 3 groups, which was a consequence of infection and not treatment. Plant-derived cysteine proteinases are therefore prime candidates for development as novel drugs for the treatment of GI nematode infections.

Phosphorylation of STAT-1 Serine 727 Is Prolonged in HLA-B27-Expressing Human Monocytic Cells

PubMed Central

Ruuska, Marja; Sahlberg, Anna S.; Granfors, Kaisa; Penttinen, Markus A.

2013-01-01

A tissue antigen, HLA-B27, is strongly associated with a group of rheumatic diseases called spondyloarthritides. Despite the intensive research, the exact role of HLA-B27 in the pathogenesis of these diseases is still unclear. Here we studied whether HLA-B27 modulates the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) serine 727 residue and the localization of STAT-1 in Salmonella-infected human monocytic cells. In addition, we studied the role of signaling molecule double-stranded RNA activated protein kinase (PKR) in these modulatory effects. U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. The PMA-differentiated cells were infected with S. enteritidis. Western blotting was used to detect the phosphorylation of STAT-1, and to visualize the localization of STAT-1 in the cells confocal microscopy was used. Specific inhibitors were employed to study the role of PKR in STAT-1 phosphorylation. We discovered that the phosphorylation of STAT-1 serine 727 is prolonged in cells expressing misfolding forms of HLA-B27 after S. enteritidis infection, whereas in mock cells and in cells expressing mutated, non-misfolding HLA-B27 the phosphorylation of serine 727 is transient. Interestingly, STAT-1 serine 727 phosphorylation is partly dependent on PKR. In addition, more STAT-1 is localized in the nucleus of HLA-B27-expressing cells, even before an external trigger, when compared to mock cells. In conclusion, our results show that the phosphorylation of STAT-1 serine 727 residue is prolonged in HLA-B27-expressing monocyte-macrophage U937 cells after bacterial infection. This is of interest since the phosphorylation of serine 727 on STAT-1 is suggested to contribute to macrophage activation and promote inflammatory responses. Therefore, our results provide a mechanism which explains how the expression of an HLA-B27 molecule can impact the course of Salmonella infection and reactive arthritis. PMID:23349666

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

PubMed

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cationic ferritin uptake by cultured anterior pituitary cells treated with the proteinase inhibitor, BOC-DPhe-Phe-Lys-H.

PubMed

Gaál, G; Bácsy, E; Rappay, G

1988-01-01

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, BOC-DPhe-Phe-Lys-H. The addition of this tripeptide aldehyde decreased the in vitro release of prolactin to 25% of the control value, while the release of growth hormone in the same cultures decreased to 33% of the control value. Prolactin immunostaining was stronger in semithin sections of proteinase-inhibitor-treated cultures than in control sections. After 2 h treatment with the inhibitor, prolactin- and growth hormone-containing secretory granules were numerous, and the number of crinophagic vacuoles had increased. In the presence of the inhibitor, the overall cytoarchitecture of parenchymal cells was well preserved, and the pathway of the uptake of cationic ferritin appeared to be unaffected.

Crystal structure of the human adenovirus proteinase with its 11 amino acid cofactor.

PubMed Central

Ding, J; McGrath, W J; Sweet, R M; Mangel, W F

1996-01-01

The three-dimensional structure of the human adenovirus-2 proteinase complexed with its 11 amino acid cofactor, pVIc, was determined at 2.6 A resolution by X-ray crystallographic analysis. The fold of this protein has not been seen before. However, it represents an example of either subtly divergent or powerfully convergent evolution, because the active site contains a Cys-His-Glu triplet and oxyanion hole in an arrangement similar to that in papain. Thus, the adenovirus proteinase represents a new, fifth group of enzymes that contain catalytic triads. pVIc, which extends a beta-sheet in the main chain, is distant from the active site, yet its binding increases the catalytic rate constant 300-fold for substrate hydrolysis. The structure reveals several potential targets for antiviral therapy. Images PMID:8617222

A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers.

PubMed

Marchiaro, Patricia; Ballerini, Viviana; Spalding, Tamara; Cera, Gabriela; Mussi, María A; Morán-Barrio, Jorgelina; Vila, Alejandro J; Viale, Alejandro M; Limansky, Adriana S

2008-08-01

The dissemination of metallo and serine carbapenem-hydrolysing beta-lactamases among Gram-negative nosocomial bacteria represents an acute problem worldwide. Here, we present a rapid and sensitive assay for the characterization of carbapenemase producers to aid in infection control and prevention. The assay involves a rapid disruption of bacterial isolates with silicon dioxide microbeads, followed by the testing in cell-free extracts of hydrolytic activity towards various beta-lactams including two carbapenems (imipenem and meropenem) and a cephalosporin (ceftazidime). A parallel testing of the effects of selective beta-lactamase inhibitors such as EDTA and clavulanic acid allows differentiation of metallo carbapenemases from serine carbapenemases, and also clavulanic-acid-sensitive from -resistant enzymes among the latter. The efficiency of bacterial disruption using silicon dioxide microbeads was identical to that of ultrasonic treatment. The subsequent microbiological assay aimed to evaluate both substrate specificity and inhibitor profile of carbapenem-hydrolysing enzymes present in the extracts and allowed an accurate differentiation of A, B and D types, as judged by the analysis of 24 well-characterized clinical strains that included metallo-beta-lactamase producers (i.e. VIM-, IMP- and SPM-type Pseudomonas producers; an L1 Stenotrophomonas maltophilia producer; and a GOB-18 Elizabethkingia meningoseptica producer) as well as serine carbapenemase producers (i.e. an SME-type Serratia marcescens producer, a GES-2 Pseudomonas aeruginosa producer, Klebsiella pneumoniae and Citrobacter freundii KPC-2 producers and OXA-type Acinetobacter baumannii producers). We have developed a convenient microbiological assay aimed to more accurately and in a short time characterize carbapenem-hydrolysing enzymes produced by Gram-negative bacteria. The assay possesses broad applicability in the clinical setting.

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

PubMed

Sheikh, Faruk G; Mukhopadhyay, Sudit S; Gupta, Prabhakar

2002-02-01

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.

Cotinine administration improves impaired cognition in the mouse model of Fragile X syndrome.

PubMed

Pardo, Marta; Beurel, Eleonore; Jope, Richard S

2017-02-01

Cotinine is the major metabolite of nicotine and has displayed some capacity for improving cognition in mouse models following chronic administration. We tested if acute cotinine treatment is capable of improving cognition in the mouse model of Fragile X syndrome, Fmr1 -/- knockout mice, and if this is related to inhibition by cotinine treatment of glycogen synthase kinase-3β (GSK3β), which is abnormally active in Fmr1 -/- mice. Acute cotinine treatment increased the inhibitory serine-phosphorylation of GSK3β and the activating phosphorylation of AKT, which can mediate serine-phosphorylation of GSK3β, in both wild-type and Fmr1 -/- mouse hippocampus. Acute cotinine treatment improved cognitive functions of Fmr1 -/- mice in coordinate and categorical spatial processing, novel object recognition, and temporal ordering. However, cotinine failed to restore impaired cognition in GSK3β knockin mice, in which a serine9-to-alanine9 mutation blocks the inhibitory serine phosphorylation of GSK3β, causing GSK3β to be hyperactive. These results indicate that acute cotinine treatment effectively repairs impairments of these four cognitive tasks in Fmr1 -/- mice, and suggest that this cognition-enhancing effect of cotinine is linked to its induction of inhibitory serine-phosphorylation of GSK3. Taken together, these results show that nicotinic receptor agonists can act as cognitive enhancers in a mouse model of Fragile X syndrome and highlight the potential role of inhibiting GSK3β in mediating the beneficial effects of cotinine on memory. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76.

PubMed

Di Bartolo, Vincenzo; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D; Acuto, Oreste

2007-03-19

The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

Phosphorylation of p53 modifies sensitivity to ionizing radiation.

PubMed

Okaichi, Kumio; Nose, Kanako; Kotake, Takako; Izumi, Nanaka; Kudo, Takashi

2011-06-01

Phosphorylation is an important modification involved in the control of p53 activity. We examined the relationship between p53 phosphorylation and cell radiosensitivity. We prepared H1299 cells (p53-null) with various mutations of p53 at three sites (serine 15, 20 and 46) and examined the radiosensitivity of the cells. In three mutant forms of p53--S15A, S20A and S46A--serine was converted to alanine at these sites to prevent phosphorylation, and in two other mutant forms, S15D and S20D, serine was converted to aspartic acid to mimic phosphorylation. H1299 cells were more radioresistant than cells with wild-type p53. Cells with the S15A and S46A mutant forms of p53 were radiosensitive, whereas those with the S15D, S20A and S20D forms showed medium radiosensitivity. Thus the sensitivity of cells to ionizing radiation varies according to the site of phosphorylation of p53.

Plasma proteins in the acquired denture pellicle enhance substrate surface free energy and Candida albicans phospholipase and proteinase activities.

PubMed

Custodio, William; Silva, Wander J; Paes Leme, Adriana F; Cury, Jaime A; Del Bel Cury, Altair A

2015-11-01

The objective of the present study was to determine if blood plasma proteins could change the proteome of the acquired denture pellicle by label-free quantitative proteomics. As pellicle proteome modulates the interaction between substrates and Candida cells, we investigated its effect on the surface free energy (SFE) of the coated resin and on Candida albicans phospholipase and aspartyl proteinase activities. Poly(methylmethacrylate) discs were exposed to saliva (control) or saliva enriched with blood plasma (experimental group). The pellicle proteome was analyzed by mass spectrometry coupled with liquid chromatography. SFE was determined by acid-base technique. After biofilm formation, phospholipase and proteinase activities were determined accordingly to classic plate methods. Data were analyzed by two-way anova and Tukey test (P < 0.05). α-Amylase, cystatins, mucins, and host-immune system proteins were the main proteins identified in the control group. Fibrinogen and albumin were observed only in the experimental group. Coated discs of the experimental group presented an increased SFE (P < 0.05). For both enzymes tested, the experimental group showed higher proteolytic activity (P < 0.001). Blood plasma changes the proteome of the acquired denture pellicle, increasing surface free energy and the activity of Candida albicans phospholipase and aspartyl proteinase. © 2014 Wiley Publishing Asia Pty Ltd.

Higher accumulation of proteinase inhibitors in flowers than leaves and fruits as a possible basis for differential feeding preference of Helicoverpa armigera on tomato (Lycopersicon esculentum Mill, Cv. Dhanashree).

PubMed

Damle, Mrunal S; Giri, Ashok P; Sainani, Mohini N; Gupta, Vidya S

2005-11-01

Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation.

PubMed

Nieminen, Mikko T; Listyarifah, Dyah; Hagström, Jaana; Haglund, Caj; Grenier, Daniel; Nordström, Dan; Uitto, Veli-Jukka; Hernandez, Marcela; Yucel-Lindberg, Tülay; Tervahartiala, Taina; Ainola, Mari; Sorsa, Timo

2018-02-06

Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and α-1-antichymotrypsin, as well as complement C1q. Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.