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1

The cysteine proteinases of the pineapple plant.  

UK PubMed Central (United Kingdom)

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

Rowan AD; Buttle DJ; Barrett AJ

1990-03-01

2

Developing novel anthelmintics from plant cysteine proteinases  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock.

Behnke Jerzy M; Buttle David J; Stepek Gillian; Lowe Ann; Duce Ian R

2008-01-01

3

The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins. Results We have performed an evolutionary comparative analysis of cysteine proteinases C1A and C13 and their putative inhibitors in representative species of different taxonomic groups that appeared during the evolution of the Viridiplantae. The results indicate that whereas C1A cysteine proteinases are present in all taxonomic groups, cystatins and C13 cysteine proteinases are absent in some basal groups. Moreover, gene duplication events have been associated to the increasing structural and functional complexities acquired in land plants. Conclusion Comparative genomic analyses have provided us valuable insights into the conservation and evolution of the cystatin inhibitory family and their putative targets, the cysteine proteinases from families C1A and C13. Functionality of both families of proteins in plants must be the result of a coevolutionary process that might have occurred during the evolution of basal and land plants leading to a complex functional relationship among them.

Martinez Manuel; Diaz Isabel

2008-01-01

4

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro.  

UK PubMed Central (United Kingdom)

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.

Stepek G; Buttle DJ; Duce IR; Lowe A; Behnke JM

2005-02-01

5

Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation.  

UK PubMed Central (United Kingdom)

Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.

Andrade SS; Silva MC; Gouvea IE; Kondo MY; Juliano MA; Sampaio MU; Oliva ML

2012-04-01

6

Baupain, a plant cysteine proteinase that hinders thrombin-induced human platelet aggregation.  

Science.gov (United States)

Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1. PMID:22185503

Andrade, Sheila S; Silva, M C C; Gouvea, I E; Kondo, M Y; Juliano, M A; Sampaio, M U; Oliva, Maria Luzia

2012-04-01

7

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.  

UK PubMed Central (United Kingdom)

SUMMARY Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1-/- and cpi-2-/-) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

Phiri AM; DE Pomerai D; Buttle DJ; Behnke JM

2013-09-01

8

Wheat cysteine proteinase gene and its application  

UK PubMed Central (United Kingdom)

The present invention obtains drought resisting related candidate EST via screening on wheat cDNA library, and clones drought resisting related cysteine proteinase gene TaCP (Triticum aestivum cystein proteinase) by means of RACE and RT-PCR technology. The present invention also relates to TaCP gene and its application. The gene is induced to express by various outer stress factors, such as drought, low temperature, salt and alkali, damage, etc. and can promote cell death to produce defence. The cloning of the gene is significant in breeding drought resisting wheat variety and in the research of wheat drought resisting molecular biology.

JING RUILIAN; ZANG QINGWEI; GUO ZHI AN

9

The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases.  

UK PubMed Central (United Kingdom)

Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests.

Visal S; Taylor MA; Michaud D

1998-09-01

10

The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases.  

Science.gov (United States)

Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests. PMID:9742962

Visal, S; Taylor, M A; Michaud, D

1998-09-01

11

[Cysteine proteinase inhibitors from soy seeds  

UK PubMed Central (United Kingdom)

Protein inhibitors of cysteine proteinases possessing unusual properties have been found in soya (Glycine max) seeds. One of the inhibitor forms has also been detected in Bowman-Birk inhibitor preparations (both commercial and purified by affinity chromatography on chymotrypsin-Sepharose ones). A peculiarity of the inhibitors is that they irreversibly lose their activity in the presence of reducing agents; therefore their effects are normally unobserved under standard conditions of cysteine proteinase inhibitor assays. Soybean inhibitors are represented by two forms with pI of 5.9 and 3.2. The molecular mass of the inhibitor whose pI is equal to 5.8 is about 14 kDa. Both inhibitors suppress the activity of papain, ficin and bromelain.

Zimacheva AV; Mosolov VV

1995-01-01

12

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).  

Science.gov (United States)

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems. PMID:15842197

Gholizadeh, Ashraf; Santha, Ittiaparambu Mana; Kohnehrouz, Bahram Baghban; Lodha, Madan Lal; Kapoor, Harish Chander

2005-12-01

13

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).  

UK PubMed Central (United Kingdom)

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems.

Gholizadeh A; Santha IM; Kohnehrouz BB; Lodha ML; Kapoor HC

2005-12-01

14

Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases.  

UK PubMed Central (United Kingdom)

Plant legumains are cysteine proteinases putatively involved in processing endogenous proteins. Phytocystatins (PhyCys) have been described as plant inhibitors of papain-like cysteine proteinases. Some PhyCys contain a carboxy terminal extension with an amino acid motif (SNSL) similar to that involved in the inhibition of legumain-like proteins by human cystatins. The role of these carboxy terminal extended PhyCys as inhibitors of legumain-like cysteine proteinases is here shown by in vitro inhibition of human legumain and legumain-like activities from barley extracts. Moreover, site-directed mutagenesis has demonstrated that the asparagine of the SNSL motif is essential in this inhibition. We prove for first time the existence of legumain inhibitors in plants.

Martinez M; Diaz-Mendoza M; Carrillo L; Diaz I

2007-06-01

15

Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya).  

UK PubMed Central (United Kingdom)

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3' untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.

McKee RA; Adams S; Matthews JA; Smith CJ; Smith H

1986-07-01

16

Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases.  

Science.gov (United States)

Plant legumains are cysteine proteinases putatively involved in processing endogenous proteins. Phytocystatins (PhyCys) have been described as plant inhibitors of papain-like cysteine proteinases. Some PhyCys contain a carboxy terminal extension with an amino acid motif (SNSL) similar to that involved in the inhibition of legumain-like proteins by human cystatins. The role of these carboxy terminal extended PhyCys as inhibitors of legumain-like cysteine proteinases is here shown by in vitro inhibition of human legumain and legumain-like activities from barley extracts. Moreover, site-directed mutagenesis has demonstrated that the asparagine of the SNSL motif is essential in this inhibition. We prove for first time the existence of legumain inhibitors in plants. PMID:17543305

Martinez, Manuel; Diaz-Mendoza, Mercedes; Carrillo, Laura; Diaz, Isabel

2007-05-25

17

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.  

UK PubMed Central (United Kingdom)

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Melo FR; Mello MO; Franco OL; Rigden DJ; Mello LV; Genú AM; Silva-Filho MC; Gleddie S; Grossi-de-Sá MF

2003-09-01

18

Identification, classification and expression pattern analysis of sugarcane cysteine proteinases  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes (more) proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB) como pertencentes à sub-sub-classe E.C.3.4.22, foram usadas na busca de clusters no banco de dados do SUCEST (SUgarCane EST project), utilizando-s o programa T-BLAST-n. Homologia de seqüências foram encontradas com 76 clusters que correspondem a prováveis proteinases cisteínicas. O alinhamento destas seqüências com a de outras proteases cisteínicas, de diversas origens, forneceu informação quanto à classificação e possível função das proteinases de cana-de-açúcar. Além disso, o padrão de expressão de cada gene foi postulado a partir da correlação direta com as bibliotecas de cDNA do SUCEST dos quais os clusters foram derivados. Uma vez que nenhum gene de protease cisteínica foi anteriormente evidenciado em cana-de-açúcar, este estudo representa uma etapa inicial para o estudo de novos aspectos bioquímicos, fisiológicos e biotecnológicos destas enzimas. Abstract in english Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinase (more) s, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST) data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.

Correa, Gustavo Coelho; Margis-Pinheiro, Márcia; Margis, Rogério

2001-12-01

19

Use of a cysteine proteinase from Carica candamarcensis as a protective agent during DNA extraction  

Directory of Open Access Journals (Sweden)

Full Text Available We describe the use of a plant cysteine proteinase isolated from latex of Carica candamarcensis as a protective agent during isolation of bacterial DNA following growth in culture of these cells. Between 100 to 720 units of proteinase (1 µg = 6 units) afforded good DNA protection when incubated with various kinds of microorganisms. Agarose gel electrophoresis showed that the resulting DNA was similar in size to DNA preparations obtained by treatment with proteinase K. The viability of the resulting material was checked by PCR amplification using species-specific primers. After standing at room temperature (25oC) for 35 days, the enzyme lost 10% of its initial activity. The enzyme stability and good yield of DNA suggest the use of this proteinase as an alternative to proteinase K.

Genelhu M.S.; Zanini M.S.; Veloso I.F.; Carneiro A.M.D.; Lopes M.T.P.; Salas C.E.

1998-01-01

20

[Cathepsin cysteine proteinases in the lung  

UK PubMed Central (United Kingdom)

Proteolytic enzymes play an important role during remodeling and digestion of extracellular matrix proteins. An overproduction of extracellular matrix or insufficient extracellular matrix digestion may result in fibrosis. Enhanced proteolytic activity or an insufficient inhibitory potential could be followed by emphysema development. Since the first reports showed an emphysema induction in rats after intratracheal application of the cysteine protease papain, a number of proteolytic enzymes involved in the remodeling of the extracellular matrix of the lung were discovered. Most of them are cysteine-, metallo-, serine- or aspartic proteases. In this paper some new findings concerning the expression, function and regulation of the activity of papain-like cysteine proteases in the process of tissue destruction and remodeling in the lung are reviewed. The functional relationship between cathepsins and other proteolytic enzymes are discussed.

Bühling F; Gerber A; Ansorge S; Welte T

1999-08-01

 
 
 
 
21

Ananain: a novel cysteine proteinase found in pineapple stem.  

UK PubMed Central (United Kingdom)

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.

Rowan AD; Buttle DJ; Barrett AJ

1988-11-01

22

Purification and characterization of a lily cysteine proteinase inhibitor  

UK PubMed Central (United Kingdom)

A cysteine proteinase inhibitor that inhibted the activity of papain was found in the bulb of lily. this CPI was purified by a series of procedures including extraction, heating, papain-Sepharose 4B affinity chromagraphy, and gel filtration on Sephadex G-100. the purified lily inhibitor was a single polypeptide with a molecular weight 12 000 in SDS-PAGE and 12 500 in gel filtration. the lily CPI was very stable below 100??and between pH2-12. It contained 0.307% neutral sugar, with Ile as the N-terminal amion acid.

Zeng Zhongkui; Yang Yuhong; Bao Jingku; Zhou Hong; Luo Liang; Deng Junlin

1996-01-01

23

[Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor].  

Science.gov (United States)

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases. PMID:21553491

Wang, Gaofeng; Peng, Deliang; Sun, Jianhua; Huang, Wenkun; Peng, Huan; Long, Haibo

2011-01-01

24

[Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor].  

UK PubMed Central (United Kingdom)

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.

Wang G; Peng D; Sun J; Huang W; Peng H; Long H

2011-01-01

25

New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases.  

UK PubMed Central (United Kingdom)

The molecular evolution of asparagine-specific cysteine proteinases, called legumains, from plants and animals was analyzed using newly available related amino acid sequences from lower eukaryotes, bacteria and Archaea. The results suggest that genuine legumains originate from prokaryote pro-legumains. The evolutionary roots of genuine legumains from plants and animals descend from Parabasalia and Alveolata before developing into their respective separate branches headed by Chlorophyta and Placozoa. The branch of legumain-like plant/animal glycosylphosphatidyl inositol transamidases separated from the general evolutionary stem of legumains at the level of lower eukaryotes. Modeling of the 3D structure of a plant genuine legumain underlined the previously suggested similarity of the active site geometry of legumains with caspases, which are Asp-specific bacterial and eukaryote proteinases.

Shutov AD; Blattner FR; Kakhovskaya IA; Müntz K

2012-02-01

26

New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases.  

Science.gov (United States)

The molecular evolution of asparagine-specific cysteine proteinases, called legumains, from plants and animals was analyzed using newly available related amino acid sequences from lower eukaryotes, bacteria and Archaea. The results suggest that genuine legumains originate from prokaryote pro-legumains. The evolutionary roots of genuine legumains from plants and animals descend from Parabasalia and Alveolata before developing into their respective separate branches headed by Chlorophyta and Placozoa. The branch of legumain-like plant/animal glycosylphosphatidyl inositol transamidases separated from the general evolutionary stem of legumains at the level of lower eukaryotes. Modeling of the 3D structure of a plant genuine legumain underlined the previously suggested similarity of the active site geometry of legumains with caspases, which are Asp-specific bacterial and eukaryote proteinases. PMID:22196948

Shutov, Andrei D; Blattner, Frank R; Kakhovskaya, Irina A; Müntz, Klaus

2011-12-22

27

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

Directory of Open Access Journals (Sweden)

Full Text Available Abstract 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.

Lepelley Maud; Amor Mohamed; Martineau Nelly; Cheminade Gerald; Caillet Victoria; McCarthy James

2012-01-01

28

A recombinant form of chagasin from Trypanosoma cruzi: inhibitory activity on insect cysteine proteinases.  

UK PubMed Central (United Kingdom)

BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg(-1) retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5-30 g kg(-1)) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.

Dos Santos Monteiro AC; de Oliveira Neto OB; Del Sarto RP; de Magalhães MT; Lima JN; Lacerda AF; Oliveira RS; Scharfstein J; da Silva MC; Valencia JW; Jiménez AV; Grossi-de-Sa MF

2008-07-01

29

Molecular cloning and expression of the gene encoding a cysteine proteinase of Spirometra erinacei.  

Science.gov (United States)

A cDNA library constructed from plerocercoid of Spirometra erinacei (SEP) was immunoscreened using rabbit anti-plerocercoid proteinase polyclonal antibody. A 1.0-kb cDNA clone encoding a cysteine proteinase composed of 336 amino acids was isolated. The amino acid sequence predicted from the cDNA showed significant homology with human and mouse cathepsin L. N-terminal amino acid sequence of the native cysteine proteinase extracted from SEP was the same as that of mature proteinase predicted from the cloned gene. The gene encoding the proteinase was characterized by Southern and Northern blot analysis using the cDNA as a probe. The proteinase with a molecular mass of 34 kDa was demonstrated in in vitro translation products using anti-proteinase polyclonal antibody. A fusion protein derived from the cDNA synthesized by Escherichia coli (TB1) using the expression vector, pMAL-c2 was identified as an immunodominant antigen by epitope-selection method and had no cross-reactivity with other parasite-infected sera. A genomic DNA library derived from SEP was screened by the colony hybridization technique using the cDNA probe. A gene with 4.5 kb encoding the proteinase was obtained, which comprised three exons and two introns. PMID:8919991

Liu, D W; Kato, H; Nakamura, T; Sugane, K

30

Molecular cloning and expression of the gene encoding a cysteine proteinase of Spirometra erinacei.  

UK PubMed Central (United Kingdom)

A cDNA library constructed from plerocercoid of Spirometra erinacei (SEP) was immunoscreened using rabbit anti-plerocercoid proteinase polyclonal antibody. A 1.0-kb cDNA clone encoding a cysteine proteinase composed of 336 amino acids was isolated. The amino acid sequence predicted from the cDNA showed significant homology with human and mouse cathepsin L. N-terminal amino acid sequence of the native cysteine proteinase extracted from SEP was the same as that of mature proteinase predicted from the cloned gene. The gene encoding the proteinase was characterized by Southern and Northern blot analysis using the cDNA as a probe. The proteinase with a molecular mass of 34 kDa was demonstrated in in vitro translation products using anti-proteinase polyclonal antibody. A fusion protein derived from the cDNA synthesized by Escherichia coli (TB1) using the expression vector, pMAL-c2 was identified as an immunodominant antigen by epitope-selection method and had no cross-reactivity with other parasite-infected sera. A genomic DNA library derived from SEP was screened by the colony hybridization technique using the cDNA probe. A gene with 4.5 kb encoding the proteinase was obtained, which comprised three exons and two introns.

Liu DW; Kato H; Nakamura T; Sugane K

1996-02-01

31

Inhibition of cysteine proteinases by autolytic digestion is mediated by CBP2/Hsp47.  

Science.gov (United States)

CBP2/Hsp47 is a glycoprotein normally limited to the ER-Golgi where it is first associated with procollagen chains at a very early point during translation of nascent chains and later with properly folded procollagen. Although CBP2/Hsp47 is regarded as a molecular chaperone belonging to the serpin superfamily, this protein does not appear to inhibit serine proteinases. Here we demonstrate that CBP2/Hsp47 functions in a manner similar to other serpin superfamily members by cross class inhibiting cysteine proteinases. A CBP2/Hsp47 to cathepsin L inactivation stoichiometery of approximately 1.5 revealed concurrent cleavage of CBP2/Hsp47 with proteinase inactivation. Cleavage of the CBP2/Hsp47 was shown to occur outside the P1-P1' at the P16-P15 and P2'-P3' bonds. In addition, the proteinase bands in SDS/PAGE diminished on reaction of the enzyme with CBP2/Hsp47. These results sustain a mechanism advocated by Bjork et al. (1998), in which cysteine proteinases assault a peptide bond in the reactive site loop of serpins, (CBP2/Hsp47) adjacent to the P1-P1' bonds involved in serine proteinase inhibition. The reaction proceeds with the substrate pathway dominating in the cysteine proteinase reaction. In these complexes the cysteine proteinases, papain and cathepsin L, are rendered more susceptible to proteolysis and are degraded by active enzyme. These properties help explain the mechanism by which CBP2/Hsp47 increases the fidelity of collagen production. Moreover, if CBP2/Hsp47 is shown to involve the multiplexin subclass of collagens, it may further provide a mechanism by which the motogen and angiogenic properties during development and/or neoplasia are regulated. PMID:12685865

Siavash, Hessam; Lopes, Marcio; Norris, Kathleen; Hebert, Carla; Nikitakis, Nikolaos; Sauk, John J

2002-01-01

32

Inhibition of cysteine proteinases by autolytic digestion is mediated by CBP2/Hsp47.  

UK PubMed Central (United Kingdom)

CBP2/Hsp47 is a glycoprotein normally limited to the ER-Golgi where it is first associated with procollagen chains at a very early point during translation of nascent chains and later with properly folded procollagen. Although CBP2/Hsp47 is regarded as a molecular chaperone belonging to the serpin superfamily, this protein does not appear to inhibit serine proteinases. Here we demonstrate that CBP2/Hsp47 functions in a manner similar to other serpin superfamily members by cross class inhibiting cysteine proteinases. A CBP2/Hsp47 to cathepsin L inactivation stoichiometery of approximately 1.5 revealed concurrent cleavage of CBP2/Hsp47 with proteinase inactivation. Cleavage of the CBP2/Hsp47 was shown to occur outside the P1-P1' at the P16-P15 and P2'-P3' bonds. In addition, the proteinase bands in SDS/PAGE diminished on reaction of the enzyme with CBP2/Hsp47. These results sustain a mechanism advocated by Bjork et al. (1998), in which cysteine proteinases assault a peptide bond in the reactive site loop of serpins, (CBP2/Hsp47) adjacent to the P1-P1' bonds involved in serine proteinase inhibition. The reaction proceeds with the substrate pathway dominating in the cysteine proteinase reaction. In these complexes the cysteine proteinases, papain and cathepsin L, are rendered more susceptible to proteolysis and are degraded by active enzyme. These properties help explain the mechanism by which CBP2/Hsp47 increases the fidelity of collagen production. Moreover, if CBP2/Hsp47 is shown to involve the multiplexin subclass of collagens, it may further provide a mechanism by which the motogen and angiogenic properties during development and/or neoplasia are regulated.

Siavash H; Lopes M; Norris K; Hebert C; Nikitakis N; Sauk JJ

2002-01-01

33

Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity  

Energy Technology Data Exchange (ETDEWEB)

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.

Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

1988-06-01

34

Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).  

Science.gov (United States)

Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6?g/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality. PMID:23830694

Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

2013-07-03

35

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal.  

Science.gov (United States)

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients. PMID:10097390

Xu, F X; Chye, M L

1999-02-01

36

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal.  

UK PubMed Central (United Kingdom)

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.

Xu FX; Chye ML

1999-02-01

37

Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: purification and enzyme kinetic properties.  

UK PubMed Central (United Kingdom)

The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papain-Sepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research.

Yadav VK; Chhikara N; Gill K; Dey S; Singh S; Yadav S

2013-08-01

38

Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide gel electrophoresis.  

Science.gov (United States)

The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne. PMID:8874065

Michaud, D; Cantin, L; Bonadé-Bottino, M; Jouanin, L; Vrain, T C

1996-08-01

39

Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytic activity but inhibits phagocytosis.  

UK PubMed Central (United Kingdom)

Inhibition of most of the expression of the cysteine proteinases of Entamoeba histolytica strain HM-1:IMSS was successfully performed by transcription of ehcp5 antisense RNA using the promoter of ehg34, which encodes a L21 ribosomal protein of E. histolytica. We have generated a stable transfectant in which the overall level of cysteine proteinase activity is strongly reduced ( 90%). This transfectant has a normal growth rate in Diamond's TYI-S-33 medium, a cytopathic and haemolytic activity similar to the control HM-1:IMSS pEhAct-Neo transfectant but with a significantly lower phagocytic activity.

Ankri S; Stolarsky T; Mirelman D

1998-05-01

40

Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance.  

UK PubMed Central (United Kingdom)

Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.

Zhang X; Liu S; Takano T

2008-09-01

 
 
 
 
41

The TvLEGU-1, a Legumain-Like Cysteine Proteinase, Plays a Key Role in Trichomonas vaginalis Cytoadherence  

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The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type ...

Rendón-Gandarilla, Francisco Javier; Ramón-Luing, Lucero de los Angeles; Ortega-López, Jaime; Rosa de Andrade, Ivone

42

Crystal structure of gingipain R: an Arg-specific bacterial cysteine proteinase with a caspase-like fold.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Gingipains are cysteine proteinases acting as key virulence factors of the bacterium Porphyromonas gingivalis, the major pathogen in periodontal disease. The 1.5 and 2.0 A crystal structures of free and D-Phe-Phe-Arg-chloromethylketone-inhibited gingipain R reveal a 435-residue, single-polypeptide c...

Eichinger, A; Beisel, H G; Jacob, U; Huber, R; Medrano, F J; Banbula, A; Potempa, J; Travis, J; Bode, W

43

Serine or cysteine proteinase inhibitor clade H (SERPHINH1) as a marker for detection of gastric cancer  

UK PubMed Central (United Kingdom)

Disclosed is a method for detecting gastric cancer, comprising: (a) providing a biological sample and (b) detecting over-expression of a GTM family member in said sample, wherein the GTM family member is serine or cysteine proteinase inhibitor clade H ("SERPINH1").

GUILFORD PARRY JOHN; HOLYOAKE ANDREW JOHN

44

Fumor invasion and metastasis resisting function and use of venin cysteine proteinase inhibitor  

UK PubMed Central (United Kingdom)

The present invention discloses the tumor invasion and metastasis resisting function and application of venin cysteine proteinase inhibitor, and belongs to the field of biomedicine. Through designing and synthesizing sv-Cystatin cDNA according to 99 amino acid sequences of Chinese cobra venin Cystatin protein, cloning to pPICZ alphaA vector and transforming Pichia yeast, stable and high expression engineering bacterium GS115-sv-Cystatin is screened out. Through further inducing expression and purification, the extracorporeal bioactivity experiment shows that the recombinant sv-Cystatin protein has the functions of inhibiting the activity of papain and inhibiting the tumor cell invasion and metastasis obviously, and so do the intracorporeal experiment. The present invention also constitutes pcDNA-sv-Cystatin eukaryotic expression vector. The present invention has huge application foreground in preventing and treating tumor.

LIN JIANYIN LIN

45

A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae).  

UK PubMed Central (United Kingdom)

A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-L-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM.

Moczo? T

2011-03-01

46

Quantification of kininogens in plasma. A functional method based on the cysteine proteinase inhibitor activity.  

UK PubMed Central (United Kingdom)

We have previously reported on a microassay based on human kininogens as cysteine proteinase inhibitors (CPIs), which could quantify partially purified kininogens from different biological fluids (J Pharmacol Meth 26, 113-124, 1991). In the present study we describe a functional method that, when assuming a 1:1 stoichiometry between papain and kininogen, allows a direct measurement of the molar concentration of kininogens in plasma. The principle of the method is that the target enzyme papain is inhibited by kininogens present in added diluted plasma. The residual activity of papain, not inhibited in this reaction, subsequently hydrolyzes the added peptide substrate (S-2302), generating a yellow colour which is read in a microplate reader at 405 nm. Relating the test samples to a standard curve established from known concentrations of E-64 (a selective low molecular weight inhibitor of cysteine proteinases), we could quantify kininogens on a molar basis. A major problem when first applying this method to plasma, was the interference of alpha 2-macroglobulin, which inhibited papain and generated a complex able to split the chromogenic substrate. The interference of alpha 2-macroglobulin was eliminated by an initial acid treatment of plasma followed by dilution with a buffer containing methylamine. The specificity for kininogens in this assay is demonstrated by the following observations: Commercial pooled normal plasma contained 3.2 microM CPI activity, in good agreement with the expected molar concentration of kininogens. After gel filtration of a plasma sample with a CPI activity of 3.4 microM, two peaks with CPI activity were identified as H-kininogen (0.9 microM) and L-kininogen (2.5 microM), both in good accordance with expected concentrations of the two kininogens. Plasma deficient of kininogens had a minimal inhibitory capacity towards papain.

Karlsrud TS; Buø L; Aasen AO; Johansen HT

1996-05-01

47

Biochemical and PMF MALDI-TOF analyses of two novel papain-like plant proteinases.  

Science.gov (United States)

Two cysteine endopeptidases from latex of Araujia angustifolia (araujiain aI and araujiain aIII) were purified and characterized by means of conventional and proteomics techniques (MALDI-TOF). N-terminal sequences showed a high percentage of identity with cysteine proteinases belonging to the papain family. The peptide mass fingerprint analysis demonstrated a close homology among both proteinases. PMID:20001923

Obregón, W D; Liggieri, C S; Morcelle, S R; Trejo, S A; Avilés, F X; Priolo, N S

2009-01-01

48

Biochemical and PMF MALDI-TOF analyses of two novel papain-like plant proteinases.  

UK PubMed Central (United Kingdom)

Two cysteine endopeptidases from latex of Araujia angustifolia (araujiain aI and araujiain aIII) were purified and characterized by means of conventional and proteomics techniques (MALDI-TOF). N-terminal sequences showed a high percentage of identity with cysteine proteinases belonging to the papain family. The peptide mass fingerprint analysis demonstrated a close homology among both proteinases.

Obregón WD; Liggieri CS; Morcelle SR; Trejo SA; Avilés FX; Priolo NS

2009-01-01

49

Action of plant proteinase inhibitors on enzymes of physiopathological importance.  

Science.gov (United States)

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. PMID:19722028

Oliva, Maria Luiza V; Sampaio, Misako U

2009-09-01

50

Activation of progelatinase A by mammalian legumain, a recently discovered cysteine proteinase.  

UK PubMed Central (United Kingdom)

The activation of progelatinase A to gelatinase A requires cleavage of an asparaginyl bond to form the N-terminus of the mature enzyme. We have asked whether the activation can be mediated by legumain, the recently discovered lysosomal cysteine proteinase that is specific for hydrolysis of asparaginyl bonds. Addition of purified legumain to the concentrated conditioned medium from HT1080 cell culture that contained both progelatinases A and B caused the conversion of the 72 kDa progelatinase A to the 62 kDa form. The progelatinase B in the medium was unaffected. Incubation of recombinant progelatinase A with legumain resulted in an almost instantaneous activation as judged by the fluorometric assay with a specific gelatinase A substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Legumain also activated progelatinase A when it was in complex with TIMP-2. Zymographic analysis and N-terminal sequencing revealed that legumain cleaved the 72 kDa progelatinase A at the bonds between Asn109-Tyr110 or Asn111-Phe112 to produce the 62 kDa mature enzyme, and that further cleavage at Asn430 also occurred to generate a 36 kDa active form. More 62 kDa gelatinase A was detected in cultures of C13 cells that over-expressed legumain than in those of the control HEK293 cells. We conclude that legumain is clearly capable of processing progelatinase A to the active enzyme in vitro and in cultured cells.

Chen JM; Fortunato M; Stevens RA; Barrett AJ

2001-05-01

51

Role of cysteine proteinase of Entamoeba histolytica in target cell death.  

UK PubMed Central (United Kingdom)

The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3-6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.

Singh D; Naik SR; Naik S

2004-08-01

52

Action of plant proteinase inhibitors on enzymes of physiopathological importance  

Directory of Open Access Journals (Sweden)

Full Text Available 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.Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

Maria Luiza V. Oliva; Misako U. Sampaio

2009-01-01

53

Action of plant proteinase inhibitors on enzymes of physiopathological importance  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, ru (more) fa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos. Abstract in english 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 inhibit (more) or 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.

Oliva, Maria Luiza V.; Sampaio, Misako U.

2009-09-01

54

Sequence conservation in the chagasin family suggests a common trend in cysteine proteinase binding by unrelated protein inhibitors.  

UK PubMed Central (United Kingdom)

The recently described inhibitor of cysteine proteinases from Trypanosoma cruzi, chagasin, was found to have close homologs in several eukaryotes, bacteria and archaea, the first protein inhibitors of cysteine proteases in prokaryotes. These previously uncharacterized 110-130 residue-long proteins share a well-conserved sequence motif that corresponds to two adjacent beta-strands and the short loop connecting them. Chagasin-like proteins also have other conserved, mostly aromatic, residues, and share the same predicted secondary structure. These proteins adopt an all-beta fold with eight predicted beta-strands of the immunoglobulin type. The phylogenetic distribution of the chagasins generally correlates with the presence of papain-like cysteine proteases. Previous studies have uncovered similar trends in cysteine proteinase binding by two unrelated inhibitors, stefin and p41, that belong to the cystatin and thyroglobulin families, respectively. A hypothetical model of chagasin-cruzipain interaction suggests that chagasin may dock to the cruzipain active site in a similar manner with the conserved NPTTG motif of chagasin forming a loop that is similar to the wedge structures formed at the active sites of papain and cathepsin L by stefin and p41.

Rigden DJ; Mosolov VV; Galperin MY

2002-08-01

55

Identification of proteinaceous inhibitors of a cysteine proteinase (an Arg-specific gingipain) from Porphyromonas gingivalis in rice grain, using targeted-proteomics approaches.  

UK PubMed Central (United Kingdom)

Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases.

Taiyoji M; Shitomi Y; Taniguchi M; Saitoh E; Ohtsubo S

2009-11-01

56

Identification of proteinaceous inhibitors of a cysteine proteinase (an Arg-specific gingipain) from Porphyromonas gingivalis in rice grain, using targeted-proteomics approaches.  

Science.gov (United States)

Porphyromonas gingivalis is known to be a major etiologic agent in the onset and progression of chronic periodontitis. Among various virulence factors that this bacterium produces, Arg- and Lys-specific cysteine proteinases (gingipains) are believed to be major determinants of the pathogenicity of P. gingivalis. Here, we report on our finding that there are inhibitors of these cysteine proteinases in a rice protein fraction. Comprehensive affinity chromatography and MS analyses resulted in the identification of 17 Arg-gingipain (Rgp)-interacting proteins in the rice endosperm. Of these, four proteins (i.e., a 26 kDa globulin, a plant lipid transfer/trypsin-alpha amylase inhibitor, the RA17 seed allergen, and an alpha amylase/trypsin inhibitor) were estimated to account for 90% of the Rgp inhibitory activity in the rice protein fraction, using a two-dimensional gel system of double-layer reverse zymography. In addition, a synthetic peptide derived from an Rgp-interacting protein, cyanate hydratase, could inhibit the growth of P. gingivalis and showed inhibitory activity against both the Arg- and Lys-gingipains. These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases. PMID:19691286

Taiyoji, Mayumi; Shitomi, Yasuyuki; Taniguchi, Masayuki; Saitoh, Eiichi; Ohtsubo, Sadami

2009-11-01

57

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.  

Science.gov (United States)

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia. PMID:11773053

Touz, María C; Nores, María J; Slavin, Ileana; Carmona, Carlos; Conrad, John T; Mowatt, Michael R; Nash, Theodore E; Coronel, Carlos E; Luján, Hugo D

2001-12-28

58

The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia.  

UK PubMed Central (United Kingdom)

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.

Touz MC; Nores MJ; Slavin I; Carmona C; Conrad JT; Mowatt MR; Nash TE; Coronel CE; Luján HD

2002-03-01

59

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds.  

UK PubMed Central (United Kingdom)

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.

Lim CO; Lee SI; Chung WS; Park SH; Hwang I; Cho MJ

1996-01-01

60

Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmic reticulum-derived vesicle is involved in protein mobilization in germinating seeds.  

UK PubMed Central (United Kingdom)

A vacuolar cysteine proteinase, designated SH-EP, is expressed in the cotyledon of germinated Vigna mungo seeds and is responsible for the degradation of storage proteins. SH-EP is a characteristic vacuolar proteinase possessing a COOH-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In this work, immunocytochemical analysis of the cotyledon cells of germinated V. mungo seeds was performed using seven kinds of antibodies to identify the intracellular transport pathway of SH-EP from ER to protein storage vacuoles. A proform of SH-EP synthesized in ER accumulated at the edge or middle region of ER where the transport vesicle was formed. The vesicle containing a large amount of proSH-EP, termed KV, budded off from ER, bypassed the Golgi complex, and was sorted to protein storage vacuoles. This massive transport of SH-EP via KV was thought to mediate dynamic protein mobilization in the cotyledon cells of germinated seeds. We discuss the possibilities that the KDEL sequence of KDEL-tailed vacuolar cysteine proteinases function as an accumulation signal at ER, and that the mass transport of the proteinases by ER-derived KV-like vesicle is involved in the protein mobilization of plants.

Toyooka K; Okamoto T; Minamikawa T

2000-02-01

 
 
 
 
61

Mass transport of proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmic reticulum-derived vesicle is involved in protein mobilization in germinating seeds.  

Science.gov (United States)

A vacuolar cysteine proteinase, designated SH-EP, is expressed in the cotyledon of germinated Vigna mungo seeds and is responsible for the degradation of storage proteins. SH-EP is a characteristic vacuolar proteinase possessing a COOH-terminal endoplasmic reticulum (ER) retention sequence, KDEL. In this work, immunocytochemical analysis of the cotyledon cells of germinated V. mungo seeds was performed using seven kinds of antibodies to identify the intracellular transport pathway of SH-EP from ER to protein storage vacuoles. A proform of SH-EP synthesized in ER accumulated at the edge or middle region of ER where the transport vesicle was formed. The vesicle containing a large amount of proSH-EP, termed KV, budded off from ER, bypassed the Golgi complex, and was sorted to protein storage vacuoles. This massive transport of SH-EP via KV was thought to mediate dynamic protein mobilization in the cotyledon cells of germinated seeds. We discuss the possibilities that the KDEL sequence of KDEL-tailed vacuolar cysteine proteinases function as an accumulation signal at ER, and that the mass transport of the proteinases by ER-derived KV-like vesicle is involved in the protein mobilization of plants. PMID:10662772

Toyooka, K; Okamoto, T; Minamikawa, T

2000-02-01

62

Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings.  

UK PubMed Central (United Kingdom)

SH-EP is the major papain-type proteinase expressed in cotyledons of germinated Vigna mungo seeds. The proteinase possesses a KDEL sequence at the C-terminus although the mature form of SH-EP is localized in vacuoles. It has also been shown that the proform of SH-EP is accumulated at the edge or middle region of the endoplasmic reticulum, and the accumulated proSH-EP is directly transported to vacuoles via the KDEL-tailed cysteine proteinase-accumulating vesicle, KV. In this study, to address the transport machinery of proSH-EP through KV, putative receptor for proSH-EP was isolated from membrane proteins of cotyledons of V. mungo seedlings using a proSH-EP-immobilized column. The deduced amino acid sequence from cDNA to the protein revealed that the putative receptor for proSH-EP is a member of vacuolar sorting receptor, VSR, that is known to be localized in the Golgi-complex and/or clathrin coated vesicle. We carried out subcellular fractionation of cotyledon cells and subsequently conducted SDS-PAGE/immunoblotting and immunocytochemistry with anti-V. mungo VSR (VmVSR) or SH-EP antibody. The results showed that VmVSR is co-localized in the fraction of the gradient in which KV existed.

Tsuru-Furuno A; Okamoto T; Minamikawa T

2001-10-01

63

Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings.  

Science.gov (United States)

SH-EP is the major papain-type proteinase expressed in cotyledons of germinated Vigna mungo seeds. The proteinase possesses a KDEL sequence at the C-terminus although the mature form of SH-EP is localized in vacuoles. It has also been shown that the proform of SH-EP is accumulated at the edge or middle region of the endoplasmic reticulum, and the accumulated proSH-EP is directly transported to vacuoles via the KDEL-tailed cysteine proteinase-accumulating vesicle, KV. In this study, to address the transport machinery of proSH-EP through KV, putative receptor for proSH-EP was isolated from membrane proteins of cotyledons of V. mungo seedlings using a proSH-EP-immobilized column. The deduced amino acid sequence from cDNA to the protein revealed that the putative receptor for proSH-EP is a member of vacuolar sorting receptor, VSR, that is known to be localized in the Golgi-complex and/or clathrin coated vesicle. We carried out subcellular fractionation of cotyledon cells and subsequently conducted SDS-PAGE/immunoblotting and immunocytochemistry with anti-V. mungo VSR (VmVSR) or SH-EP antibody. The results showed that VmVSR is co-localized in the fraction of the gradient in which KV existed. PMID:11673621

Tsuru-Furuno, A; Okamoto, T; Minamikawa, T

2001-10-01

64

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli  

Directory of Open Access Journals (Sweden)

Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Toaldo CB; Steindel M; Sousa MA; Tavares CC

2001-01-01

65

Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70) and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despit (more) e the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

Toaldo, CB; Steindel, M; Sousa, MA; Tavares, CC

2001-01-01

66

X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis.  

UK PubMed Central (United Kingdom)

CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P4(1)2(1)2 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R (free) = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.

Gomes MT; Teixeira RD; Lopes MT; Nagem RA; Salas CE

2012-12-01

67

The families of papain- and legumain-like cysteine proteinases from embryonic axes and cotyledons of Vicia seeds: developmental patterns, intracellular localization and functions in globulin proteolysis.  

UK PubMed Central (United Kingdom)

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

Fischer J; Becker C; Hillmer S; Horstmann C; Neubohn B; Schlereth A; Senyuk V; Shutov A; Müntz K

2000-05-01

68

A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression.  

Science.gov (United States)

The barley cysteine proteinase B (EPB) is the main protease responsible for the degradation of endosperm storage proteins providing nitrogenous nutrients to support the growth of young seedlings. The expression of this enzyme is induced in the germinating seeds by the phytohormone, gibberellin, and suppressed by another phytohormone, abscisic acid. In situ hybridization experiments indicate that EPB is expressed in the scutellar epithelium within 24 h of seed germination, but the aleurone tissue surrounding the starchy endosperm eventually becomes the main tissue expressing this enzyme. The EPB gene family of barley consists of two very similar genes, EPB1 and EPB2, both of which have been mapped to chromosome 3. The sequences of EPB1 and EPB2 match with the two previously published cDNA clones indicating that both genes are expressed. Interestingly, neither of these genes contain any introns, a rare phenomenon in which all members of a small gene family are active intronless genes. Sequence comparison indicates that the barley EPB family can be classified as cathepsin L-like endopeptidases and is most closely related to two legume cysteine proteinases (Phaseolus vulgaris EP-C1 and Vigna mungo SHEP) which are also involved in seed storage protein degradation. The promoters of EPB1 and EPB2 have been linked to the coding sequence of a reporter gene, GUS, encoding beta-glucuronidase, and introduced into barley aleurone cells using the particle bombardment method. Transient expression studies indicate that EPB promoters are sufficient to confer the hormonal regulation of these genes. PMID:8756590

Mikkonen, A; Porali, I; Cercos, M; Ho, T H

1996-05-01

69

Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP).  

Science.gov (United States)

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages. PMID:10447692

Okamoto, T; Yuki, A; Mitsuhashi, N; Minamikawa, T; Mimamikawa, T

1999-08-01

70

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.  

UK PubMed Central (United Kingdom)

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important.

Ohshita T; Nikawa T; Towatari T; Katunuma N

1992-10-01

71

Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro.  

Science.gov (United States)

Intraperitoneal administration of N-(L-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-prolin e (CA-074) to rats at a dose of 4 mg/100 g greatly inhibited cathepsin-B activity in both liver and kidney for at least 4 h. Its inhibitory effect was selective for cathepsin-B activity in the liver but not in the kidney. The effects of selective inhibition of cathepsin-B activity by CA-074 treatment, and general inhibition of cysteine proteinases by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-3-methylbutylamid e (E-64-c) on the degradation of fluorescein isothiocyanate (FITC)-labeled asialofetuin in liver lysosomes, were examined in vivo. Undegraded or partially degraded FITC-labeled asialofetuin and its FITC-labeled degradation products were both found in the lysosomes and were easily separated by Sephadex G-25' column chromatography. The FITC-labeled degradation products were mainly lysine with an FITC-labeled epsilon-amino group. Accumulation of undegraded or partially degraded FITC-labeled asialofetuin in the lysosomes was marked after E-64-c treatment, but slight after CA-074 treatment. Under the marked inhibition of general lysosomal cysteine-proteinase activity by E-64-c or marked selective inhibition of cathepsin-B activity by CA-074 in vitro, degradation of FITC-labeled asialofetuin by disrupted lysosomes was analyzed on the basis of measurement of FITC-labeled degradation products by Sephadex G-25 column chromatography. It was suppressed markedly but incompletely by E-64-c as well as by CA-074, but more weakly than by E-64-c. These results shows that E-64-sensitive cysteine proteinases are important in lysosomal protein degradation, but cathepsin B has only a role in part and that an E-64-resistant proteinase(s) may also be important. PMID:1382984

Ohshita, T; Nikawa, T; Towatari, T; Katunuma, N

1992-10-01

72

Structure/function analysis of human cystatin SN and comparison of the cysteine proteinase inhibitory profiles of human cystatins C and SN.  

UK PubMed Central (United Kingdom)

Cystatins are reversible, competitive inhibitors of cysteine proteinases. Their inhibitory profiles, as well as their affinities for target enzymes, vary with different cysteine proteinases. Human cystatin C and salivary cystatin SN are 120- and 121-amino-acid (a.a.) proteins, respectively, and both contain 2 disulfide bonds. In this study, we examined the structure/function relationship of cystatin SN with respect to the inhibition of papain, with particular emphasis on the role of cystatin SN's cysteine residues, and addressed the inhibitory profiles of these two human cystatins on several cysteine proteinases (papain, clostripain, and calpain II). The full-length recombinant cystatin C and cystatin SN, and cystatin SN variants (C-truncated [C-tr; a.a. 1-102], delta 56-60 deletion, cysteine 74-->serine [C74S], cys 84-->serine [C84S], cysteine 98-->serine [C98S], and cysteine 118-->serine [C118S]) were cloned, expressed, and produced in the pET30(b) and pGEX2T Escherichia coli expression systems. All recombinant proteins were tested for the inhibition of papain, and the full-length proteins were also tested for the inhibition of clostripain and calpain II. The secondary structures of the cystatins were also determined and compared. The results showed that the full-length cystatin C and cystatin SN, and the cystatin SN variants C98S and C118S inhibited the activity of papain. However, cystatin SN C-tr and delta 56-60 variants exhibited no inhibitory activity toward papain, while the cystatin SN variants C74S and C84S exhibited slight inhibition at higher concentrations. These results suggested that in the inhibition of papain by cystatin SN, the first disulfide loop is more important than the second. In addition, cystatin C, but not cystatin SN, inhibited calpain II, while neither cystatin inhibited clostripain, and these results, in conjunction with those from other studies, indicated that cystatin C is a broader-spectrum inhibitor of cysteine proteinases than cystatin SN.

Hiltke TR; Lee TC; Bobek LA

1999-08-01

73

Comparison of clinical and environmental isolates of Acanthamoeba based on morphology, protease and gelatinase activity, and the cysteine proteinase gene  

Directory of Open Access Journals (Sweden)

Full Text Available Acanthamoeba spp. are opportunistic pathogens that cause amebic keratitis and granulomatous amebic encephalitis in man. Recent attempts to correlate pathogenicity with species have been proven difficult due to inconsistencies in morphology-based classification. The objectives of this study were: (1) to compare clinical and environmental isolates based on morphology, protease and gelatinase activity, and the cysteine proteinase (CP) gene, and (2) to determine whether these features can be used to differentiate the isolates. Results show some degree of variation in trophozoite and cyst morphology. Zymography, demonstrated gross differences in banding patterns, and the protease activity of clinical isolates was greater than the environmental isolates (p-value < 0.01). Amplification of the CP gene yielded two bands in the environmental isolates, approximately 755 bp and 440 bp in length. In contrast, only one band, either the 755 bp or 440 bp band was amplified in the clinical isolates. The results confirmed the limitations of morphology in differentiating Acanthamoeba species, and suggest that zymography, protease activity, and detection of the CP gene are useful reference tests to distinguish pathogenic from non-pathogenic isolates.

Gil M. Penuliar; Ronald R. Matias; Filipinas F. Natividad

2010-01-01

74

Biochemical characterisation of MX-4, a plant cysteine protease of broad specificity and high stability  

UK PubMed Central (United Kingdom)

A new proteinase, mexicain-IV (MX-4), has been purified to homogeneity from the latex fruits of Jacaratia mexicana (formely Pileus mexicanus), a plant member of the Caricaceae family. MX-4 shows a Mr of 23.7kDa, pI of 9.3 and maximum proteolytic activity on casein and BAPNA at pH 8.0-8.5 and pH 7.0-7.5, respectively. The amino acid sequence of MX-4 and its reversible inhibition by HgCl? show that the proteinase belongs to the family of cysteine proteinases. This enzyme exhibits rather broad substrate specificity, although there seems to be a slight preference for cleavage of peptides having certain hydrophobic residues in the P2 position. Biochemical and circular dichroism studies revealed that this enzyme belongs to the ?+? class of proteins, in agreement with the results obtained by X-ray crystallographic structure determination, and showed that MX-4 has a higher pH and thermal stability than other members of the Caricaceae family, including papain. These properties make this novel protease a suitable novel analytical tool for the proteomic analysis of peptide fragments of great potential interest in the food industry and other industries.

Oliver-Salvador MdC; Lian Z; Laursen RA; Bolaños-García VM; Soriano-García M

2011-05-01

75

MOLECULAR CLONING OF TRYPSIN-LIKE CDNAS AND COMPARISON OF PROTEINASE ACTIVITIES IN THE SALIVARY GLANDS AND GUT OF THE TARNISHED PLANT BUG LYGUS LINEOLARIS (HEMIPTERA: MIRIDAE)  

Science.gov (United States)

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseina...

76

The TvLEGU-1, a legumain-like cysteine proteinase, plays a key role in Trichomonas vaginalis cytoadherence.  

Science.gov (United States)

The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence. PMID:23509742

Rendón-Gandarilla, Francisco Javier; Ramón-Luing, Lucero de Los Angeles; Ortega-López, Jaime; Rosa de Andrade, Ivone; Benchimol, Marlene; Arroyo, Rossana

2013-01-01

77

Entamoeba histolytica cysteine proteinase 5 binds integrin on colonic cells and stimulates NFkappaB-mediated pro-inflammatory responses.  

UK PubMed Central (United Kingdom)

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10(5) deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to ?(V)?(3) integrin on Caco-2 colonic cells and stimulates NF?B-mediated pro-inflammatory responses. PCP5 RGD binding to ?(V)?(3) integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-?B essential modulator (NEMO). As NEMO is required for activation of the IKK?-IKK? complex and NF?B signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2(-/-) mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for ?(V)?(3) integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.

Hou Y; Mortimer L; Chadee K

2010-11-01

78

Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.  

Science.gov (United States)

The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor. PMID:22833537

Nogueira, Fábio C S; Silva, Carlos P; Alexandre, Daniel; Samuels, Richard I; Soares, Emanoella L; Aragão, Francisco J L; Palmisano, Giuseppe; Domont, Gilberto B; Roepstorff, Peter; Campos, Francisco A P

2012-07-26

79

Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.  

UK PubMed Central (United Kingdom)

The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

Nogueira FC; Silva CP; Alexandre D; Samuels RI; Soares EL; Aragão FJ; Palmisano G; Domont GB; Roepstorff P; Campos FA

2012-08-01

80

Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor  

DEFF Research Database (Denmark)

The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

Nogueira, Fábio C S; Silva, Carlos P

2012-01-01

 
 
 
 
81

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages.  

UK PubMed Central (United Kingdom)

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.

Lecaille F; Muno D; Kominami E; Ishidoh K

2004-06-01

82

In-silico comparative study of inhibitory mechanism of Plant Serine Proteinase Inhibitors.  

UK PubMed Central (United Kingdom)

The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use Plant Proteinase Inhibitors (PIs) gene which are used to control the proteolysis mechanism of Pest by inhibiting gut Serine Proteinase (SP). Present work investigates the utility of computer aided methods to study the mechanism of Protein-Protein interactions and thereby inhibition of Serine Proteinase by PIs. Hence 3D models of Serine Proteinase as well as Serine Proteinase Inhibitors (SPIs) generated using homology modeling. Validations of constructed models have been done by PROCHECK, VERIFY3D, ERRAT and PROSA. Prediction of Protein interacting surface patches and site specific protein docking was performed by using ZDOCK Server. Backbone refinement of output protein complexes was executed in Fiber Dock server. Interaction study between SP and SPIs complexes shows their comparative inhibition efficacy, measured in terms of number of hydrogen bonds, Van dar wall attraction and docking energy. This work reported that Vigna marina and Phaseolus oligospermus are having better inhibition efficiency in comparison to other inhibitors.

Siva Prasad CV; Gupta S; Gaponenko A; Dhar M

2012-01-01

83

EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Abstract Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human...

Allhorn Maria; Olsén Arne; Collin Mattias

84

Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa1  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57Bl/6 mice, fastuosain and bromelain injected intraperitoneally were protective, and very few nodules of B16F10-Nex2 melanoma ce...

Guimarães-Ferreira, Carla A; Rodrigues, Elaine G; Mortara, Renato A; Cabral, Hamilton; Serrano, Fabiana A; Ribeiro-dos-Santos, Ricardo

85

Parasite cysteine proteinase interactions with alpha 2-macroglobulin or kininogens: differential pathways modulating inflammation and innate immunity in infection by pathogenic trypanosomatids.  

UK PubMed Central (United Kingdom)

Plasma extravasation is a common endothelium response to tissue injury provoked by pathogens. Herein I will review studies showing that host proteinase inhibitors (e.g., alpha2-macroglobulin (alpha2M) or kininogens) interact with protozoan cysteine proteinases (CPs) in extravascular infection sites, linking inflammation to innate immunity by different mechanisms. Using human monocytes as antigen presenting cells, we first demonstrated that alpha2M entrapment of cruzipain, a Trypanosoma cruzi CP, reduced the activation threshold of cruzipain-specific CD4 T cells due to facilitated uptake of alpha2M-cruzipain complexes by the multiscavenger receptor (CD91). More recently, studies of the mechanisms underlying inflammation elicited by T. cruzi revealed that kininogens, once bound to glycosaminoglycans, are not able to efficiently inactivate cruzipain via their inhibitory cystatin-like domains. Instead, we found that cruzipain readily processes surface-bound kininogens, liberating bioactive kinins. Acting as paracrine hormones, kinins vigorously activate host cells through bradykinin (BK) receptors, thus stimulating endocytic uptake of the pathogen. Rather than unilaterally enhancing parasite infectivity, the liberated kinins activate innate immunity by potently stimulating dendritic cell maturation via the BK B2 receptor. The discovery of chagasin, a novel family of endogenous inhibitors expressed by trypanosomatids, is likely another regulatory player involved in the dynamics of the inflammatory response.

Scharfstein J

2006-01-01

86

Potential value of a rice protein extract, containing proteinaceous inhibitors against cysteine proteinases from Porphyromonas gingivalis, for managing periodontal diseases.  

UK PubMed Central (United Kingdom)

Arg-specific gingipain (Rgp) is a major pathogenic determinant of Porphyromonas gingivalis which is a major pathogen in periodontal disease. We prepared protein extracts with Rgp-inhibitory activity from polished rice (Oryza sativa) and evaluated the effects of these extracts on the growth and pathogenicity of P. gingivalis. The extracts inhibited the proteolytic degradation of human proteins by P. gingivalis proteinases, and repressed the growth and homotypic biofilm formation of P. gingivalis. The disruption of adhesion of epithelial cells by P. gingivalis was also restricted by the rice protein extracts. Our results suggested that the rice protein extracts suppressed the pathogenicity and growth of P. gingivalis by inhibiting the bacterial proteinase activities, implying that the Rgp-inhibitory proteins prepared from rice may be potentially valuable as nutraceutical agents for preventing periodontal diseases.

Taiyoji M; Yamanaka T; Tsuno T; Ohtsubo S

2013-01-01

87

Potential value of a rice protein extract, containing proteinaceous inhibitors against cysteine proteinases from Porphyromonas gingivalis, for managing periodontal diseases.  

Science.gov (United States)

Arg-specific gingipain (Rgp) is a major pathogenic determinant of Porphyromonas gingivalis which is a major pathogen in periodontal disease. We prepared protein extracts with Rgp-inhibitory activity from polished rice (Oryza sativa) and evaluated the effects of these extracts on the growth and pathogenicity of P. gingivalis. The extracts inhibited the proteolytic degradation of human proteins by P. gingivalis proteinases, and repressed the growth and homotypic biofilm formation of P. gingivalis. The disruption of adhesion of epithelial cells by P. gingivalis was also restricted by the rice protein extracts. Our results suggested that the rice protein extracts suppressed the pathogenicity and growth of P. gingivalis by inhibiting the bacterial proteinase activities, implying that the Rgp-inhibitory proteins prepared from rice may be potentially valuable as nutraceutical agents for preventing periodontal diseases. PMID:23291749

Taiyoji, Mayumi; Yamanaka, Takashi; Tsuno, Takuo; Ohtsubo, Sadami

2013-01-07

88

Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.  

UK PubMed Central (United Kingdom)

The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings.

Taneyama M; Okamoto T; Yamane H; Minamikawa T

2001-11-01

89

Involvement of gibberellins in expression of a cysteine proteinase (SH-EP) in cotyledons of Vigna mungo seedlings.  

Science.gov (United States)

The expression of a papain-type proteinase, designated SH-EP, in cotyledons of Vigna mungo seedlings has been shown to require some factors in the embryonic axes. Gibberellin A1 (GA(1)) and GA(20) were identified by GC-MS in embryonic axes of V. mungo seedlings. The level of accumulation of SH-EP in cotyledons of V. mungo seedlings was greatly reduced by treatment of the seeds with uniconazole-P, an inhibitor for GA biosynthesis. The reduced level of accumulation of SH-EP in cotyledons by uniconazole-P was recovered by exogenous application of GA(1) and GA(20) to the seedlings. PMID:11726715

Taneyama, M; Okamoto, T; Yamane, H; Minamikawa, T

2001-11-01

90

Cysteine protease enhances plant-mediated bollworm RNA interference.  

UK PubMed Central (United Kingdom)

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

Mao YB; Xue XY; Tao XY; Yang CQ; Wang LJ; Chen XY

2013-09-01

91

Cysteine protease enhances plant-mediated bollworm RNA interference.  

Science.gov (United States)

Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects. PMID:23460027

Mao, Ying-Bo; Xue, Xue-Yi; Tao, Xiao-Yuan; Yang, Chang-Qing; Wang, Ling-Jian; Chen, Xiao-Ya

2013-03-04

92

Delivery of a cocktail DNA vaccine encoding cysteine proteinases type I, II and III with solid lipid nanoparticles potentiate protective immunity against Leishmania major infection.  

UK PubMed Central (United Kingdom)

Earlier generations of Leishmania vaccines have reached the third-phase of clinical trials, however none of them have shown adequate efficacy due to lack of an appropriate adjuvant. In this study, cationic solid lipid nanoparticles (cSLNs) were used to formulate three pDNAs encoding L. major cysteine proteinase type I (cpa), II (cpb) and III (cpc). BALB/c mice were immunized twice with a 3-week interval, with SLN-pcDNA-cpa/b/c, pcDNA-cpa/b/c, SLN, SLN-pcDNA and PBS. Footpad assessments, parasite burden, cytokine and antibody responses were evaluated. Mice vaccinated with SLN-pcDNA-cpa/b/c significantly (p<0.05) showed higher protection levels with specific Th1 immune response development compared to other groups. This is the first report demonstrating cSLNs as a nanoscale vehicle boosting immune response quality and quantity; in a designable trend. The nanomedical feature of this novel formulation can be applied for wide-spread use in genetic vaccination against leishmaniasis, which is currently managed only through relatively ineffectual therapeutic regimens.

Doroud D; Zahedifard F; Vatanara A; Najafabadi AR; Taslimi Y; Vahabpour R; Torkashvand F; Vaziri B; Rafati S

2011-07-01

93

Cysteine proteinase type I, encapsulated in solid lipid nanoparticles induces substantial protection against Leishmania major infection in C57BL/6 mice.  

UK PubMed Central (United Kingdom)

Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C-terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL/6 mice. Therefore, SLN-CPB and SLN-CPB(-CTE) formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen-specific T-helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN-?, IgG2a and lower IL-4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN-?/IL-4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN-CPB(-CTE) vaccinated group, post-challenge. Thus, SLN-CPB(-CTE) was shown to induce specific Th1 immune responses to control L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells.

Doroud D; Zahedifard F; Vatanara A; Najafabadi AR; Rafati S

2011-06-01

94

Delivery of a cocktail DNA vaccine encoding cysteine proteinases type I, II and III with solid lipid nanoparticles potentiate protective immunity against Leishmania major infection.  

Science.gov (United States)

Earlier generations of Leishmania vaccines have reached the third-phase of clinical trials, however none of them have shown adequate efficacy due to lack of an appropriate adjuvant. In this study, cationic solid lipid nanoparticles (cSLNs) were used to formulate three pDNAs encoding L. major cysteine proteinase type I (cpa), II (cpb) and III (cpc). BALB/c mice were immunized twice with a 3-week interval, with SLN-pcDNA-cpa/b/c, pcDNA-cpa/b/c, SLN, SLN-pcDNA and PBS. Footpad assessments, parasite burden, cytokine and antibody responses were evaluated. Mice vaccinated with SLN-pcDNA-cpa/b/c significantly (p<0.05) showed higher protection levels with specific Th1 immune response development compared to other groups. This is the first report demonstrating cSLNs as a nanoscale vehicle boosting immune response quality and quantity; in a designable trend. The nanomedical feature of this novel formulation can be applied for wide-spread use in genetic vaccination against leishmaniasis, which is currently managed only through relatively ineffectual therapeutic regimens. PMID:21530597

Doroud, Delaram; Zahedifard, Farnaz; Vatanara, Alireza; Najafabadi, Abdolhossein Rouholamini; Taslimi, Yasaman; Vahabpour, Rouholah; Torkashvand, Fatemeh; Vaziri, Behrooz; Rafati, Sima

2011-04-19

95

Cysteine proteinase type I, encapsulated in solid lipid nanoparticles induces substantial protection against Leishmania major infection in C57BL/6 mice.  

Science.gov (United States)

Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C-terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL/6 mice. Therefore, SLN-CPB and SLN-CPB(-CTE) formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen-specific T-helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN-?, IgG2a and lower IL-4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN-?/IL-4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN-CPB(-CTE) vaccinated group, post-challenge. Thus, SLN-CPB(-CTE) was shown to induce specific Th1 immune responses to control L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells. PMID:21410716

Doroud, D; Zahedifard, F; Vatanara, A; Najafabadi, A R; Rafati, S

2011-06-01

96

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism  

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Full Text Available Abstract Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn ?-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide.

Phakthanakanok Krongsakda; Ratanakhanokchai Khanok; Kyu Khin; Sompornpisut Pornthep; Watts Aaron; Pinitglang Surapong

2009-01-01

97

Cysteine-based redox regulation and signaling in plants.  

UK PubMed Central (United Kingdom)

Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signaling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs) are disulfide bonds, sulfenic acids, S-glutathione adducts, S-nitrosothiols and to a lesser extent S-sulfenyl-amides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.

Couturier J; Chibani K; Jacquot JP; Rouhier N

2013-01-01

98

Cysteine-based redox regulation and signaling in plants  

Science.gov (United States)

Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signaling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs) are disulfide bonds, sulfenic acids, S-glutathione adducts, S-nitrosothiols and to a lesser extent S-sulfenyl-amides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.

Couturier, Jeremy; Chibani, Kamel; Jacquot, Jean-Pierre; Rouhier, Nicolas

2013-01-01

99

Effects of plant species switching on dynamics of amylase and proteinase activity of Bemisia tabaci biotype B and Trialeurodes vaporariorum  

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Full Text Available Bemisia tabaci biotype B (Bt) and Trialeurodes vaporariorum (Tv) are two whiteflies that often coexist on greenhouse-grown vegetable crops in northern China. The host plant species of B. tabaci biotypeB have been increasing with the spread of its invaded areas. To clarify the effects of plant species on the performances of the two species, amylase and proteinase activity dynamics of both whitefly species were evaluated when their host plant species switched from tomato (preferred by both species, also used as the control) to cotton (preferred by Bt and suitable for Tv), cabbage (preferred by Bt, but unsuitable for Tv) or maize (unsuitable for both species) plants. There were no prominent effects of plant species switching on the amylase activity of the two whitefly species. The amylase activity of Bt was 1.49–1.66 folds higher than that of Tv when fed on tomato or when switched to different host species. There were no effects on the proteinase activity of Bt when switched to a different host species, but that of Tv decreased by 29.9–42.7%. The proteinase activity of Bt was 1.30 and 1.21 folds higher than that of Tv when switched to cabbage and cotton plants, respectively. There were no interspecific differences in proteinase activity when switched to maize plants. Bt amylase activity dynamics were expressed as activation then inhibition or inhibition then activationwhen switched to cabbage and maize plants; however, those of Tv were smooth. Amylase activity dynamics were similar for both species when switched to cotton. Although proteinase activity dynamics of Bt were expressed as activation then inhibition when switched to any of the three plant species tested, the activation period was longer when switched to maize plants. In the case of Tv, proteinase activity dynamics were similarly smooth regardless of which plant species switched to. Our results indicated that Bt was more adaptable to plant species switching than T. vaporariorum.

Guifen Zhang; Fang Lei; Fanghao Wan; Jun Ma; Yuguo Yang

2008-01-01

100

Characterization of cysteine proteases in Malian medicinal plants.  

UK PubMed Central (United Kingdom)

Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed.

Bah S; Paulsen BS; Diallo D; Johansen HT

2006-09-01

 
 
 
 
101

Characterization of cysteine proteases in Malian medicinal plants.  

Science.gov (United States)

Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed. PMID:16621376

Bah, Sékou; Paulsen, Berit S; Diallo, Drissa; Johansen, Harald T

2006-03-22

102

EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity.  

UK PubMed Central (United Kingdom)

BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSION: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

Allhorn M; Olsén A; Collin M

2008-01-01

103

EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity  

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Full Text Available Abstract Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. Results We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. Conclusion We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

Allhorn Maria; Olsén Arne; Collin Mattias

2008-01-01

104

Cationic solid lipid nanoparticles loaded by cysteine proteinase genes as a novel anti-leishmaniasis DNA vaccine delivery system: characterization and in vitro evaluations.  

UK PubMed Central (United Kingdom)

PURPOSE: Leishmaniasis is a major health problem in many tropical and sub-tropical countries and development of a safe and easily-available vaccine has high priority. Although several antigens potentially capable of inducing protective immunity have been studied, in the absence of pharmaceutical industry interest they have remained as fine publications only. Amongst them, Cathepsin L-like cysteine proteinases (CPs) have received considerable attention and type I and II CPs have been used in a heterologous prime-boost vaccination regime for experimental visceral leishmaniasis in dogs. Due to the promising results of the mentioned vaccination regime, we aimed to evaluate cationic solid lipid nanoparticles (cSLNs) for in vitro delivery of cpa, cpb and cpb(CTE) intended to be used as a cocktail DNA vaccine in our forthcoming studies. METHODS: cSLNs were formulated of cetyl palmitate, cholesterol, DOTAP and Tween 80 via melt emulsification method followed by high shear homogenization. Different formulations were prepared by anchoring pDNAs on the surface of cSLNs via charge interaction. The formulations were characterized according to their size and zeta potential as well as pDNA integrity and stability against DNase I treatment. Lipoplexes' cytotoxicity was investigated on COS-7 cells by MTT test. The effect of the DOTAP:pDNA ratio on protection ability and cytotoxicity was also studied. In vitro transfection efficiency was qualified by fluorescent microscopy and quantified using flow cytometry technique. RESULTS: cSLN-pDNA complexes were formulated with suitable size and zeta potential. Efficiency/cytotoxicity ratio of cSLN-pDNAs formulations was comparable to linear PEI-25KD-pDNAs polyplexes while exhibiting significantly lower cytotoxicity. CONCLUSION: Tested formulations were able to deliver immunogenic CP genes efficiently. This data proves the ability of this system as a promising DNA vaccine carrier for leishmaniasis to cover the main drawback of naked pDNA delivery that is rapid elimination from the circulation.

Doroud D; Vatanara A; Zahedifard F; Gholami E; Vahabpour R; Rouholamini Najafabadi A; Rafati S

2010-01-01

105

Silencing of cystatin M in metastatic oral cancer cell line MDA-686Ln by siRNA increases cysteine proteinases and legumain activities, cell proliferation and in vitro invasion.  

UK PubMed Central (United Kingdom)

Cystatins are inhibitors of lysosomal cysteine proteinases. Cystatin M demonstrates more diverse tissue distribution, target specificity and biological function than other cystatins from the same family. We utilized small interference RNAs (siRNA) to silence cystatin M gene expression in a metastatic oral cancer cell line (MDA-686Ln) that expresses a high level of cystatin M. We tested four different siRNAs targeted to different sites of the cystatin M mRNA, and found three out of the four siRNAs were effective in suppressing cystatin M expression by >50% at both mRNA and protein levels, as measured by quantitative real-time RT-PCR and Western blotting. We used siRNA-#1, which demonstrated highest efficiency of silencing cystatin M, to evaluate the phenotypic outcome of silencing cystatin M in MDA-686Ln cells. Cystatin M inhibition significantly increased the enzymatic activities of cathepsins B and L and legumain while reducing cysteine protease inhibitor activity both in the media and intracellularly. MDA-686Ln cells treated with siRNA#1 demonstrated markedly increased proliferation rate, in vitro motility and Matrigel invasiveness. Collectively, our data show that silencing of cystatin M in tumor cells not only increases their invasion and motility via cysteine-proteinase-dependent pathways, but also renders them hyperproliferative through a currently unknown mechanism.

Vigneswaran N; Wu J; Nagaraj N; James R; Zeeuwen P; Zacharias W

2006-01-01

106

Cysteine protease mcII-Pa executes programmed cell death during plant embryogenesis  

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Programmed cell death (PCD) is indispensable for eukaryotic development. In animals, PCD is executed by the caspase family of cysteine proteases. Plants do not have close homologues of caspases but possess a phylogenetically distant family of cysteine proteases named metacaspases. The cellular funct...

Bozhkov, Peter V.; Suarez, Maria F.; Filonova, Lada H.; Daniel, Geoffrey; Zamyatnin, Andrey A.; Rodriguez-Nieto, Salvador

107

The cysteine regulatory complex from plants and microbes: what was old is new again.  

UK PubMed Central (United Kingdom)

The physical organization of enzymes in metabolism is an old concept being revisited by new experimental approaches. In plants and microbes, the enzymes of cysteine biosynthesis-serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS)-form a bi-enzyme complex called the cysteine regulatory complex (CRC), which likely plays a role in modulating cysteine biosynthesis in response to sulfur nutrient state. Structural and biochemical studies of SAT and OASS as individual enzymes and recent advances in structural, biophysical, and in vivo analysis of the CRC provide new insights on the function of this macromolecular assembly in plants and microbes and opens biotechnology and pharmaceutical opportunities for future exploration.

Jez JM; Dey S

2013-04-01

108

Aspartic proteinases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco.  

UK PubMed Central (United Kingdom)

Carnivorous plants acquire significant amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes accumulates acidic fluid containing aspartic proteinase (AP) in its trapping organs (pitchers), suggesting that the plant utilizes insect protein as a nitrogen source. Aspartic proteinases have been purified and characterized from sterile pitcher fluid of several species of Nepenthes; however, there is, as of yet, no information about sequence and expression of Nepenthes AP genes. To identify the pitcher AP, we cloned plant AP homologs from N. alata and examined their expressions. Five AP homologs ( NaAP1-NaAP5) were obtained by reverse transcription-polymerase chain reaction with degenerate primers designed for the conserved sequences of plant APs. Alignment of deduced amino acid sequences with other plant APs demonstrated that NaAP1-NaAP4 contained a plant-specific insert (PSI), a unique sequence of plant AP. However, NaAP5 did not possess the insert, and had a shorter sequence (by >100 amino acids) than the other APs. Northern analysis using a part of the coding region of NaAP1 as a probe showed that bands of approx. 1.8 kb corresponding to the sizes of NaAP1-NaAP4 mRNA were present in roots, stems, leaves, tendrils, and lower part of the pitchers, but a band of approx. 1.3 kb corresponding to the size of NaAP5 mRNA was not observed in any organs. In pitchers, highest expressions of NaAP1-NaAP4 were seen in the lower part of open pitchers containing natural prey, suggesting that the expressions of NaAP1-NaAP4 are coupled with prey capture. Transcripts of NaAP2 and NaAP4 were detected in the digestive glands, where AP secretion may occur. This result suggests that NaAP2 and NaAP4 are the possible APs secreted into the pitcher of N. alata.

An CI; Fukusaki E; Kobayashi A

2002-03-01

109

Aspartic proteinases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco.  

Science.gov (United States)

Carnivorous plants acquire significant amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes accumulates acidic fluid containing aspartic proteinase (AP) in its trapping organs (pitchers), suggesting that the plant utilizes insect protein as a nitrogen source. Aspartic proteinases have been purified and characterized from sterile pitcher fluid of several species of Nepenthes; however, there is, as of yet, no information about sequence and expression of Nepenthes AP genes. To identify the pitcher AP, we cloned plant AP homologs from N. alata and examined their expressions. Five AP homologs ( NaAP1-NaAP5) were obtained by reverse transcription-polymerase chain reaction with degenerate primers designed for the conserved sequences of plant APs. Alignment of deduced amino acid sequences with other plant APs demonstrated that NaAP1-NaAP4 contained a plant-specific insert (PSI), a unique sequence of plant AP. However, NaAP5 did not possess the insert, and had a shorter sequence (by >100 amino acids) than the other APs. Northern analysis using a part of the coding region of NaAP1 as a probe showed that bands of approx. 1.8 kb corresponding to the sizes of NaAP1-NaAP4 mRNA were present in roots, stems, leaves, tendrils, and lower part of the pitchers, but a band of approx. 1.3 kb corresponding to the size of NaAP5 mRNA was not observed in any organs. In pitchers, highest expressions of NaAP1-NaAP4 were seen in the lower part of open pitchers containing natural prey, suggesting that the expressions of NaAP1-NaAP4 are coupled with prey capture. Transcripts of NaAP2 and NaAP4 were detected in the digestive glands, where AP secretion may occur. This result suggests that NaAP2 and NaAP4 are the possible APs secreted into the pitcher of N. alata. PMID:11882933

An, Chung-Il; Fukusaki, Ei-ichiro; Kobayashi, Akio

2001-10-16

110

In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors.  

UK PubMed Central (United Kingdom)

We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera.

Parde VD; Sharma HC; Kachole MS

2010-09-01

111

C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase.  

Science.gov (United States)

Sulfhydryl-endopeptidase (SH-EP) is a papain-type vacuolar proteinase expressed in cotyledons of germinated Vigna mungo seeds, and the enzyme possesses a C-terminal propeptide containing KDEL tail, an endoplasmic reticulum retention signal for soluble proteins. SH-EP is transported to vacuoles via a KDEL vesicle (KV) through a Golgi complex-independent route. To see the function of the KDEL sequence of SH-EP, wild-type SH-EP and its KDEL deletion mutant (SH-EPDeltaKDEL) were heterologously expressed in Arabidopsis and in cultured tobacco Bright Yellow 2 cells, and their intracellular transport pathways and localizations were analyzed. A combination of the results from analyses for transformed Arabidopsis and tobacco (Nicotiana tabacum) cells indicated that wild-type SH-EP is packed into KV-like vesicles through the KDEL sequence and is transported to vacuoles in the cells of transformants. In contrast, KV was not formed/induced in the cells expressing SH-EPDeltaKDEL, and the mutant protein was mainly secreted. Therefore, the C-terminal KDEL sequence of the KDEL-tailed cysteine proteinase is thought to be involved in the formation of KV, and in the efficient vacuolar transport of the proteins through KV. PMID:12913146

Okamoto, Takashi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Nishimura, Mikio; Minamikawa, Takao

2003-08-01

112

Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.  

UK PubMed Central (United Kingdom)

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.

Dunse KM; Stevens JA; Lay FT; Gaspar YM; Heath RL; Anderson MA

2010-08-01

113

Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field.  

Science.gov (United States)

Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor. PMID:20696895

Dunse, K M; Stevens, J A; Lay, F T; Gaspar, Y M; Heath, R L; Anderson, M A

2010-08-09

114

A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants.  

Science.gov (United States)

We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3%) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1~5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus tremula x Populus alba) and Picea sitchensis, appear to possess only a single homologue. GFP fusion experiments demonstrate that DcCDT1 and OsCDT1 are targeted to both the plant cytoplasmic membranes and cell walls. Constitutive expression of DcCDT1 or OsCDT1 confers Cd-tolerance to transgenic A. thaliana plants by lowering the accumulation of Cd in the cells. The functions of the DcCDT1 family members are discussed in the light of these findings. PMID:19816106

Matsuda, Taiki; Kuramata, Masato; Takahashi, Yoshihiro; Kitagawa, Etsuko; Youssefian, Shohab; Kusano, Tomonobu

2009-05-25

115

A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants.  

UK PubMed Central (United Kingdom)

We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3%) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1~5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus tremula x Populus alba) and Picea sitchensis, appear to possess only a single homologue. GFP fusion experiments demonstrate that DcCDT1 and OsCDT1 are targeted to both the plant cytoplasmic membranes and cell walls. Constitutive expression of DcCDT1 or OsCDT1 confers Cd-tolerance to transgenic A. thaliana plants by lowering the accumulation of Cd in the cells. The functions of the DcCDT1 family members are discussed in the light of these findings.

Matsuda T; Kuramata M; Takahashi Y; Kitagawa E; Youssefian S; Kusano T

2009-05-01

116

Papaya proteinase IV amino acid sequence.  

UK PubMed Central (United Kingdom)

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV.

Ritonja A; Buttle DJ; Rawlings ND; Turk V; Barrett AJ

1989-11-01

117

Papaya proteinase IV amino acid sequence.  

Science.gov (United States)

The amino acid sequence of papaya proteinase IV (PPIV), a major proteinase from the latex of Carica papaya [(1989) Biochem. J. 261, 469-476] is described. The enzyme has a high degree of sequence identity with papaya proteinase III, chymopapain and papain (81, 70 and 67%, respectively), and is clearly a member of the papain superfamily of cysteine proteinases. Nevertheless, the sequence shows substitution of certain residues conserved in all other known members of the superfamily. It is suggested that some of these substitutions may account for the unusual specificity of PPIV. PMID:2591528

Ritonja, A; Buttle, D J; Rawlings, N D; Turk, V; Barrett, A J

1989-11-20

118

FRACTIONATION OF DIGESTIVE PROTEINASES FROM TENEBRIO MOLITOR (COLEOPTERA: TENEBRIONIDAE) LARVAE AND ROLE IN PROTEIN DIGESTION  

Science.gov (United States)

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chro...

119

A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap).  

Science.gov (United States)

The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme. PMID:21307583

Takahashi, Kenji; Suzuki, Takehiro; Nishii, Wataru; Kubota, Keiko; Shibata, Chiaki; Isobe, Toshiaki; Dohmae, Naoshi

2011-02-07

120

A cysteine endopeptidase ("dionain") is involved in the digestive fluid of Dionaea muscipula (Venus's fly-trap).  

UK PubMed Central (United Kingdom)

The carnivorous plant Dionaea muscipula (Venus's flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS-PAGE band protein. The name "dionain" is proposed for the enzyme.

Takahashi K; Suzuki T; Nishii W; Kubota K; Shibata C; Isobe T; Dohmae N

2011-01-01

 
 
 
 
121

PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L.) manufactured from the latex of raw papaya fruits. The enzyme is able to...

Ezekiel Amri; Florence Mamboya

122

The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation.  

UK PubMed Central (United Kingdom)

BACKGROUND: Fieldwork has thoroughly established that most eggs are intensely predated. Among the few exceptions are the aerial egg clutches from the aquatic snail Pomacea canaliculata which have virtually no predators. Its defenses are advertised by the pigmented ovorubin perivitellin providing a conspicuous reddish coloration. The nature of the defense however, was not clear, except for a screening for defenses that identified a neurotoxic perivitellin with lethal effect on rodents. Ovorubin is a proteinase inhibitor (PI) whose role to protect against pathogens was taken for granted, according to the prevailing assumption. Through biochemical, biophysical and feeding experiments we studied the proteinase inhibitor function of ovorubin in egg defenses. METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry sequencing indicated ovorubin belongs to the Kunitz-type serine proteinase inhibitor family. It specifically binds trypsin as determined by small angle X-ray scattering (SAXS) and cross-linking studies but, in contrast to the classical assumption, it does not prevent bacterial growth. Ovorubin was found extremely resistant to in vitro gastrointestinal proteolysis. Moreover feeding studies showed that ovorubin ingestion diminishes growth rate in rats indicating that this highly stable PI is capable of surviving passage through the gastrointestinal tract in a biologically active form. CONCLUSIONS: To our knowledge, this is the first direct evidence of the interaction of an egg PI with a digestive protease of potential predators, limiting predator's ability to digest egg nutrients. This role has not been reported in the animal kingdom but it is similar to plant defenses against herbivory. Further, this would be the only defense model with no trade-offs between conspicuousness and noxiousness by encoding into the same molecule both the aposematic warning signal and an antinutritive/antidigestive defense. These defenses, combined with a neurotoxin and probably unpalatable factors would explain the near absence of predators, opening new perspectives in the study of the evolution and ecology of egg defensive strategies.

Dreon MS; Ituarte S; Heras H

2010-01-01

123

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.  

UK PubMed Central (United Kingdom)

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

Bienert GP; Cavez D; Besserer A; Berny MC; Gilis D; Rooman M; Chaumont F

2012-07-01

124

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor.  

Science.gov (United States)

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition. PMID:23950094

Marinho-Prado, Jeanne Scardini; Lourenção, A L; Guedes, R N C; Pallini, A; Oliveira, J A; Oliveira, M G A

2012-07-06

125

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor.  

UK PubMed Central (United Kingdom)

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48 h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.

Marinho-Prado JS; Lourenção AL; Guedes RN; Pallini A; Oliveira JA; Oliveira MG

2012-10-01

126

Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon.  

Science.gov (United States)

Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants. We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA. The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E. coli SATase. DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings. Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect. A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine. PMID:7608200

Saito, K; Yokoyama, H; Noji, M; Murakoshi, I

1995-07-01

127

Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon.  

UK PubMed Central (United Kingdom)

Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants. We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA. The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E. coli SATase. DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings. Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect. A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine.

Saito K; Yokoyama H; Noji M; Murakoshi I

1995-07-01

128

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

UK PubMed Central (United Kingdom)

ETHNOPHARMACOLOGICAL RELEVANCE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Camilo Patiño A; María Benjumea D; Andrés Pereañez J

2013-09-01

129

Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism.  

UK PubMed Central (United Kingdom)

Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca²?. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2;1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2;1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2;1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2;1, one of which could be the properties of the lipid bilayer.

Frick A; Järvå M; Ekvall M; Uzdavinys P; Nyblom M; Törnroth-Horsefield S

2013-09-01

130

Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism.  

Science.gov (United States)

Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2;1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2;1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2;1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2;1, one of which could be the properties of the lipid bilayer. PMID:23819815

Frick, Anna; Järvå, Michael; Ekvall, Mikael; Uzdavinys, Povilas; Nyblom, Maria; Törnroth-Horsefield, Susanna

2013-09-15

131

The catalytic cysteine and histidine in the plant acyl-acyl carrier protein thioesterases.  

UK PubMed Central (United Kingdom)

The plant acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of their utilities in the genetic engineering of plant seed oils. Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis, suggesting that these enzymes activate the hydrolysis of the thioester bond using the same basic catalytic machinery as those of proteases and lipases. To identify the cysteine and histidine residues that are critical in catalysis we substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine (Cys-320) to an Ala or a Ser, and three conserved histidines (His-140, His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a substantial loss of enzyme activity. However, only C320A and H285A completely inactivated the enzyme, indicating that these two residues are essential for catalysis. Considerable activity (>60%) still remained when Cys-320 was converted to a Ser, but this mutant (C320S) displayed a reversed sensitivity toward thiol or serine hydroxyl inhibitors compared with the wild-type enzyme. A pH optimal study demonstrates that while the wild-type enzyme has the highest activity between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to higher pH and a significant increase of activity around pH 12. This result suggests that Arg-285 (pKa 12) is deprotonated at high pH, thus partially mimicking the role of His-285 for proton abstraction in the wild-type enzyme. We conclude that the Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can attack the thioester bond of the substrate 12:0 ACP, assisted by His-285. Because plant TEs are highly conserved in length and sequence and the residues investigated here are completely conserved in all available TEs, it is reasonable to believe that homologues of Cys-320 and His-285 are present in the active sites of all plant acyl-ACP TEs.

Yuan L; Nelson BA; Caryl G

1996-02-01

132

The catalytic cysteine and histidine in the plant acyl-acyl carrier protein thioesterases.  

Science.gov (United States)

The plant acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) play an essential role in chain termination during de novo fatty acid synthesis and are of biochemical interest because of their utilities in the genetic engineering of plant seed oils. Biochemical data have shown the possible involvement of an active-site cysteine and a histidine in catalysis, suggesting that these enzymes activate the hydrolysis of the thioester bond using the same basic catalytic machinery as those of proteases and lipases. To identify the cysteine and histidine residues that are critical in catalysis we substituted, in a 12:0 ACP TE (Uc FatB1), a conserved cysteine (Cys-320) to an Ala or a Ser, and three conserved histidines (His-140, His-285, and His-345) to an Ala or an Arg. Each Ala mutation caused a substantial loss of enzyme activity. However, only C320A and H285A completely inactivated the enzyme, indicating that these two residues are essential for catalysis. Considerable activity (>60%) still remained when Cys-320 was converted to a Ser, but this mutant (C320S) displayed a reversed sensitivity toward thiol or serine hydroxyl inhibitors compared with the wild-type enzyme. A pH optimal study demonstrates that while the wild-type enzyme has the highest activity between pH 8.5 and 9.5, the mutant H285A shows a shifted optimum to higher pH and a significant increase of activity around pH 12. This result suggests that Arg-285 (pKa 12) is deprotonated at high pH, thus partially mimicking the role of His-285 for proton abstraction in the wild-type enzyme. We conclude that the Cys-320 of the wild-type enzyme and Ser-320 of the mutant enzyme can attack the thioester bond of the substrate 12:0 ACP, assisted by His-285. Because plant TEs are highly conserved in length and sequence and the residues investigated here are completely conserved in all available TEs, it is reasonable to believe that homologues of Cys-320 and His-285 are present in the active sites of all plant acyl-ACP TEs. PMID:8631942

Yuan, L; Nelson, B A; Caryl, G

1996-02-16

133

Activated recombinant adenovirus proteinases  

Energy Technology Data Exchange (ETDEWEB)

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.

Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

1999-08-10

134

Activated recombinant adenovirus proteinases  

Science.gov (United States)

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.

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

1999-08-10

135

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].  

UK PubMed Central (United Kingdom)

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.

Ma DM; Bai JJ; Jian Q; Lao HH; Ye X; Luo JR

2003-09-01

136

Expression of diverse midgut serine proteinases in the sericigenous Lepidoptera Antheraea assamensis (Helfer) is influenced by choice of host plant species.  

UK PubMed Central (United Kingdom)

Antheraea assamensis is reared on various species of the Lauraceae family from north-east India for its distinctive cocoon silk. We demonstrate differential expression of digestive trypsin and chymotrypsins in larvae feeding on a primary host, Persea bombycina Kosterm., in comparison to larvae feeding on Litsea monopetala Roxb. using in vitro proteolytic assays, zymogram analyses with proteinase inhibitors, restriction digestion of RNA-PCR amplicons and quantitative real-time PCR (RT-PCR). Eight novel members of the serine proteinase gene family were identified, including an intron-spliced trypsin (AaPb4) and seven putative chymotrypsins (AaPb2, AaPb4, AaPb12, AaLm4, AaLm6, AaLm19 and AaLm29). Midgut transcript levels of the putative trypsin were higher in larvae fed P. bombycina whereas levels of transcripts encoding putative chymotrypsins were higher in larvae reared on L. monopetala. Complex, differential expression of sequence divergent midgut serine proteinases may reflect the ability of lepidopteran larvae to feed on different species of host plants. Possible implications of host plant choice on the digestive physiology of A. assamensis are discussed.

Saikia M; Singh YT; Bhattacharya A; Mazumdar-Leighton S

2011-02-01

137

Selective adsorption of plant cysteine peptidases onto TiO2.  

UK PubMed Central (United Kingdom)

A crude extract rich in plant cysteine peptidases was obtained from the latex of the fruits of Araujia hortorum, a South American climbing plant. The highly concentrated extract was immobilized onto titanium dioxide to produce biocatalysts through a simple adsorption procedure. Absorbance measurement at 280 nm and Bradford's method for protein quantification revealed that the protein content of the crude extract was selectively adsorbed onto the titanium dioxide surface at a very high rate. In 5 min of contact with the support all protein present in the crude extract was selectively withdrawn from the solution, leading to an immobilized biocatalyst with a high protein concentration. Caseinolytic assays indicated that, except for the catalyst obtained with the highest crude amount contacted with the support, all the proteolytic activity present in the crude extract was adsorbed onto TiO(2). The amidasic activity of the immobilized catalysts (Ah/TiO(2)) was tested in the hydrolysis of a synthetic chromogenic substrate (PFLNA) showing partial deactivation with respect to the native enzyme. In amidasic activity assays the ionic strength of the buffer medium showed to be a key feature to consider in order to avoid protease desorption from the support, indicating the importance of electrostatic interactions between the enzymes and TiO(2). Reuse of the produced biocatalysts with PFLNA as substrate revealed that after five successive uses Ah/TiO(2) retained more than 20% of its initial activity.

Llerena-Suster CR; Foresti ML; Briand LE; Morcelle SR

2009-08-01

138

Selective adsorption of plant cysteine peptidases onto TiO2.  

Science.gov (United States)

A crude extract rich in plant cysteine peptidases was obtained from the latex of the fruits of Araujia hortorum, a South American climbing plant. The highly concentrated extract was immobilized onto titanium dioxide to produce biocatalysts through a simple adsorption procedure. Absorbance measurement at 280 nm and Bradford's method for protein quantification revealed that the protein content of the crude extract was selectively adsorbed onto the titanium dioxide surface at a very high rate. In 5 min of contact with the support all protein present in the crude extract was selectively withdrawn from the solution, leading to an immobilized biocatalyst with a high protein concentration. Caseinolytic assays indicated that, except for the catalyst obtained with the highest crude amount contacted with the support, all the proteolytic activity present in the crude extract was adsorbed onto TiO(2). The amidasic activity of the immobilized catalysts (Ah/TiO(2)) was tested in the hydrolysis of a synthetic chromogenic substrate (PFLNA) showing partial deactivation with respect to the native enzyme. In amidasic activity assays the ionic strength of the buffer medium showed to be a key feature to consider in order to avoid protease desorption from the support, indicating the importance of electrostatic interactions between the enzymes and TiO(2). Reuse of the produced biocatalysts with PFLNA as substrate revealed that after five successive uses Ah/TiO(2) retained more than 20% of its initial activity. PMID:19394803

Llerena-Suster, C R F; Foresti, M L; Briand, L E; Morcelle, S R

2009-03-27

139

Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stress responses in transgenic Arabidopsis plants.  

UK PubMed Central (United Kingdom)

In this report a full-length cDNA, SPCP2, which encoded a putative papain-like cysteine protease was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP2 contained 1101 nucleotides (366 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 68% to 83%) with plant cysteine proteases, including Actinidia deliciosa, Arabidopsis thaliana, Brassica oleracea, Phaseolus vulgaris, Pisum sativa, Vicia faba, Vicia sativa and Vigna mungo. RT-PCR analysis showed that SPCP2 gene expression was enhanced significantly in natural senescent leaves and in dark-, abscisic acid- (ABA-), jasmonic acid- (JA-) and ethephon-induced senescent leaves, but was almost not detected in mature green leaves, stems, and roots. Transgenic Arabidopsis with constitutive SPCP2 expression exhibited earlier floral transition from vegetative to reproductive growth, higher percentage of incompletely developed siliques per plant, reduced average fresh weight and lower germination percentage of seed, and higher salt and drought stress tolerance compared to those of control. Based on these results we conclude that sweet potato papain-like cysteine protease, SPCP2, is a functional senescence-associated gene, and its expression causes altered developmental characteristics and stress responses in transgenic Arabidopsis plants.

Chen HJ; Su CT; Lin CH; Huang GJ; Lin YH

2010-07-01

140

Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stress responses in transgenic Arabidopsis plants.  

Science.gov (United States)

In this report a full-length cDNA, SPCP2, which encoded a putative papain-like cysteine protease was isolated from senescent leaves of sweet potato (Ipomoea batatas). SPCP2 contained 1101 nucleotides (366 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 68% to 83%) with plant cysteine proteases, including Actinidia deliciosa, Arabidopsis thaliana, Brassica oleracea, Phaseolus vulgaris, Pisum sativa, Vicia faba, Vicia sativa and Vigna mungo. RT-PCR analysis showed that SPCP2 gene expression was enhanced significantly in natural senescent leaves and in dark-, abscisic acid- (ABA-), jasmonic acid- (JA-) and ethephon-induced senescent leaves, but was almost not detected in mature green leaves, stems, and roots. Transgenic Arabidopsis with constitutive SPCP2 expression exhibited earlier floral transition from vegetative to reproductive growth, higher percentage of incompletely developed siliques per plant, reduced average fresh weight and lower germination percentage of seed, and higher salt and drought stress tolerance compared to those of control. Based on these results we conclude that sweet potato papain-like cysteine protease, SPCP2, is a functional senescence-associated gene, and its expression causes altered developmental characteristics and stress responses in transgenic Arabidopsis plants. PMID:20129700

Chen, Hsien-Jung; Su, Cheng-Ting; Lin, Chia-Hung; Huang, Guan-Jhong; Lin, Yaw-Huei

2010-02-02

 
 
 
 
141

Production of Plant Proteinase from Jack Fruit (Artocarpus integrifolis) as a Source of Dairy Enzyme I. Isolation, Partial Purification and Some Properties  

Directory of Open Access Journals (Sweden)

Full Text Available The aim of the present work was to search for a novel plant proteinase enzyme from Jack fruit (Artocarpus integrifolis) as a source of dairy enzymes that would be natural products which can be easily extracted at relatively low cost and no legal barriers. This enzyme was subjected to a purification scheme composed of ammonium sulfate fractionation followed by gel filtration on G-100 Sephadex column. The enzyme was purified 2.70-fold with a total yield of 23.77% of the original activity. There were relationship between temperature and incubation time, the enzyme activity increase was observed up to 55°C for 60 min reaction time and still constant thereafter. Proteinase was active over a broad temperature range retained about 37.4 and 24.9% of temperature activity at 35 and 80°C for 5 and 60 min. An energy of activation of 9.98 KJ mole?1 for the enzyme activity was derived from the Arrhenius plot of initial velocity (Vo) across a temperature ranging from 40 to 55°C. The optimum pH was pH 7.5. The rate of thermal inactivation proceeded more rapidly at pH 7.0 and 8.0, when heating at 50°C for 60 min the enzyme activity lost about 95 and 92% its activity, respectively. Michaelis-constant of (Km) values of 2.0 mg ml?1 and a maximum initial velocity (Vmax) of 0.75 µ moles mg?1 when casein used as a substrate. A Molecular weight (MW) determination of ~22 kDa was estimated by gel filtration methods using a Sephadex G-100. Cu2+, K2+ , Fe2+ and Zn2+ strongly inhibited the enzyme. However, Ca++ slightly stimulated. EDTA, sodium azide, Sodium citrate and urea among the chemical reagents inhibited the proteinase activity.

Al-Sayed Al-Tanboly

2003-01-01

142

[Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation  

Science.gov (United States)

Arrowhead Proteinase Inhibitor(API), one kind of pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insects such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid pBIAH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-II were transferred into three lines of local winter wheat--JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed plants showed that three of Kmr green plants (two from 866554, one from JD-1) were PCR positive with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were carried out among selfiedoffsprings of transformed positive plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-II gene was checked via ELISA in our study, all of three PCR and Southern blot positive plants could yield high level of NPT-II. This data provided a more powerful evidence for integration of insecticide gene into wheat genome. PMID:10876664

Mu; Liu; Zhou; Wen; Zhang; Wei

1999-01-01

143

[Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation  

UK PubMed Central (United Kingdom)

Arrowhead Proteinase Inhibitor(API), one kind of pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insects such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid pBIAH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-II were transferred into three lines of local winter wheat--JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed plants showed that three of Kmr green plants (two from 866554, one from JD-1) were PCR positive with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were carried out among selfiedoffsprings of transformed positive plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-II gene was checked via ELISA in our study, all of three PCR and Southern blot positive plants could yield high level of NPT-II. This data provided a more powerful evidence for integration of insecticide gene into wheat genome.

Mu HM; Liu SJ; Zhou WJ; Wen YX; Zhang WJ; Wei RX

1999-01-01

144

Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH-EP)  

UK PubMed Central (United Kingdom)

A plant cysteine endopeptidase, designated SH-EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies. Here we show that the 43 kDa intermediate of SH-EP formed in the endoplasmic reticulum is transported to protein bodies and processed to the 33 kDa mature form during transport or thereafter, and that the COOH-terminal propeptide of 10 amino acid residues containing a KDEL sequence, which is known as a retention signal for the endoplasmic reticulum lumen, is processed to form the mature SH-EP.

Okamoto T; Nakayama H; Seta K; Isobe T; Minamikawa T

1994-08-01

145

Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH-EP)  

Science.gov (United States)

A plant cysteine endopeptidase, designated SH-EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies. Here we show that the 43 kDa intermediate of SH-EP formed in the endoplasmic reticulum is transported to protein bodies and processed to the 33 kDa mature form during transport or thereafter, and that the COOH-terminal propeptide of 10 amino acid residues containing a KDEL sequence, which is known as a retention signal for the endoplasmic reticulum lumen, is processed to form the mature SH-EP. PMID:8076688

Okamoto, T; Nakayama, H; Seta, K; Isobe, T; Minamikawa, T

1994-08-29

146

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.  

Science.gov (United States)

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases. PMID:12360523

Franco, Octávio L; Grossi de Sá, Maria F; Sales, Maurício P; Mello, Luciane V; Oliveira, Adeliana S; Rigden, Daniel J

2002-11-15

147

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.  

UK PubMed Central (United Kingdom)

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.

Franco OL; Grossi de Sá MF; Sales MP; Mello LV; Oliveira AS; Rigden DJ

2002-11-01

148

Proteinase activity regulation by glycosaminoglycans  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distribut (more) ed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

Tersariol, I.L.S.; Pimenta, D.C.; Chagas, J.R.; Almeida, P.C.

2002-02-01

149

Modes of inhibition of cysteine proteases.  

UK PubMed Central (United Kingdom)

Cysteine proteases are involved in many physiological processes and their hyperactivity may lead to severe diseases. Nature has developed various strategies to protect cells and whole organisms against undesired proteolysis. One of them is the control of proteolytic activity by inhibition. This paper presents the mechanisms underlying the action of proteinaceous inhibitors of cysteine proteinases and covers propeptides binding backwards relative to the substrate or distorting the protease catalytic centre similarly to serpins, the p35 protein binding covalently to the enzyme, and cystatins that are exosite binding inhibitors. The paper also discusses tyropins and chagasins that, although unrelated to cystatins, inhibit cysteine proteinases by a similar mechanism, as well as inhibitors of the apoptosis protein family that bind in a direction opposite to that of the substrate, similarly to profragments. Special attention is given to staphostatins, a novel family of inhibitors acting in an unusual manner.

Rzychon M; Chmiel D; Stec-Niemczyk J

2004-01-01

150

Influence of air temperature on proteinase activity and beverage quality in Coffea arabica  

Directory of Open Access Journals (Sweden)

Full Text Available Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

Hellen Marília Couto de Abreu; Paula Macedo Nobile; Milton Massao Shimizu; Paula Yuri Yamamoto; Emerson Alves Silva; Carlos Augusto Colombo; Paulo Mazzafera

2012-01-01

151

Serine and cysteine protease-like genes in the genome of a gall midge and their interactions with host plant genotypes.  

UK PubMed Central (United Kingdom)

Proteases play important roles in a wide range of physiological processes in organisms. For plant-feeding insects, digestive proteases are targets for engineering protease inhibitors for pest control. In this study, we identified 105 putative serine- and cysteine-protease genes from the genome of the gall midge Mayetiola destructor (commonly known as Hessian fly), a destructive pest of wheat. Among the genes, 31 encode putative trypsins, 18 encode putative chymotrypsins, seven encode putative cysteine proteases, and the remaining may encode either other proteases or protease homologues. Developmental stage- and tissue-specific expression profiles of the genes encoding putative trypsins, chymotrypsins, and cysteine proteases were determined by quantitative reverse-transcription PCR. Comparative analyses of stage- and tissue-specific expression patterns suggested that several genes are likely to encode digestive proteases in the M. destructor larval gut, including genes encoding putative trypsins MDP3, MDP5, MDP9, MDP24, MDP48, MDP51, MDP57, MDP61, MDP71, and MDP90; genes encoding putative chymotrypsins MDP1, MDP7, MDP8, MDP18, MDP19, and MDP20; and genes encoding putative cysteine proteases MDP95 and MDP104. The expression of some protease genes was affected by plant genotypes. Genes encoding trypsins MDP3, MDP9, and MPD23, chymotrypsins MDP20 and MDP21, and cysteine proteases MDP99 and MDP104 were upregulated in M. destructor larvae feeding in resistant plants, whereas genes encoding trypsins MDP12, MDP24, and MDP33, and chymotrypsins mdp8, mdp15, and mdp16 were downregulated in M. destructor larvae feeding in resistant plants. This study provides a foundation for further comparative studies on proteases in different insects, and further characterization of M. destructor digestive proteases and their interactions with host plants, as well as potential targets for transgenic wheat plants.

Chen H; Zhu YC; Whitworth RJ; Reese JC; Chen MS

2013-08-01

152

Analysis of pineapple [Ananas comosus (L.) Merr.] fruit proteinases by 2-D zymography and direct identification of the major zymographic spots by mass spectrometry  

UK PubMed Central (United Kingdom)

Cysteine proteinases present in pineapple plants are phytotherapeutical agents with anti-edematous, anti-inflammatory, anti-thrombotic and fibrinolytic activities. Active components involved have been only partially identified as bromelain, the major proteinase in pineapple fruits. In this work, pineapple fruit extracts were analysed by 2-D zymography. Clear spots, corresponding to enzymatic activities, were excised, digested with trypsin and submitted to MALDI-ToF mass spectrometry for enzyme identification. The most representative enzymes were identified as bromelains, including their isoforms and their post-translational modifications. The novelty of the present study is the identification of proteolytic activities by means of direct MALDI-ToF MS analysis of the zymographic spots. Enzymes were identified without the need for 2-D gel electrophoresis or purification. 2-D zymography can offer not only the complete map of the enzymes present in a biological sample or in a food matrix, but it can allow also their direct identification including their isotypes.

Larocca Marilena; Rossano Rocco; Santamaria Monica; Riccio Paolo

2010-12-01

153

Serpins in plants and green algae  

DEFF Research Database (Denmark)

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.

Roberts, Thomas Hugh; Hejgaard, JØrn

2008-01-01

154

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits.  

Science.gov (United States)

A new protease (araujiain h I) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an iso-electric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0-9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 microM. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36-48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment. PMID:10882171

Priolo, N; Morcelle del Valle, S; Arribére, M C; López, L; Caffini, N

2000-01-01

155

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits.  

UK PubMed Central (United Kingdom)

A new protease (araujiain h I) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an iso-electric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0-9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl2, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 microM. When assayed on N-alpha-CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36-48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.

Priolo N; Morcelle del Valle S; Arribére MC; López L; Caffini N

2000-01-01

156

Propeptide-Mediated Inhibition of Cognate Gingipain Proteinases  

Science.gov (United States)

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism’s cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with Ki values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Huq, N. Laila; Seers, Christine A.; Toh, Elena C. Y.; Dashper, Stuart G.; Slakeski, Nada; Zhang, Lianyi; Ward, Brent R.; Meuric, Vincent; Chen, Dina; Cross, Keith J.; Reynolds, Eric C.

2013-01-01

157

Co-factor activated recombinant adenovirus proteinases  

Energy Technology Data Exchange (ETDEWEB)

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.

Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

1996-08-06

158

Co-factor activated recombinant adenovirus proteinases  

Science.gov (United States)

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.

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

1996-08-06

159

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.  

UK PubMed Central (United Kingdom)

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment. However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity.

Liebig HD; Skern T; Luderer M; Sommergruber W; Blaas D; Kuechler E

1991-07-01

160

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.  

Science.gov (United States)

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase and an active 2A proteinase of human rhinovirus 2 was expressed, alpha complementation was not affected, as the 2A proteinase cleaved itself off the alpha fragment. However, fusion of an inactive 2A prevented alpha complementation, as the 2A polypeptide remained fused to the alpha fragment. After random mutation of the 2A gene by PCR amplification, mutants were screened; M13 phage defective in alpha complementation were obtained at an efficiency of 5% and were shown to contain mutated 2A genes. Intermolecular cleavage was then examined by expressing an alpha fragment-inactive proteinase fusion protein as substrate for an active 2A proteinase expressed from an M13 vector. alpha complementation indicated intermolecular processing of the 2A cleavage site on the alpha fragment-inactive proteinase fusion protein. This versatile system thus allows the high-density screening of both active and inactive proteinase mutants, cleaving either intramolecularly or intermolecularly, and should be applicable to other proteinases of high specificity. Images

Liebig, H D; Skern, T; Luderer, M; Sommergruber, W; Blaas, D; Kuechler, E

1991-01-01

 
 
 
 
161

Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization.  

Science.gov (United States)

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination. PMID:9346282

Becker, C; Senyuk, V I; Shutov, A D; Nong, V H; Fischer, J; Horstmann, C; Müntz, K

1997-09-01

162

Proteinases in developing dental enamel.  

UK PubMed Central (United Kingdom)

For almost three decades, proteinases have been known to reside within developing dental enamel. However, identification and characterization of these proteinases have been slow and difficult, because they are present in very small quantities and they are difficult to purify directly from the mineralizing enamel. Enamel matrix proteins such as amelogenin, ameloblastin, and enamelin are cleaved by proteinases soon after they are secreted, and their cleavage products accumulate in the deeper, more mature enamel layers, while the full-length proteins are observed only at the surface. These results suggest that proteinases are necessary for "activating" enamel proteins so the parent proteins and their cleavage products may perform different functions. A novel matrix metalloproteinase named enamelysin (MMP-20) was recently cloned from tooth tissues and was later shown to localize primarily within the most recently formed enamel. Furthermore, recombinant porcine enamelysin was demonstrated to cleave recombinant porcine amelogenin at virtually all of the sites that have previously been described in vivo. Therefore, enamelysin is at least one enzyme that may be important during early enamel development. As enamel development progresses to the later stages, a profound decrease in the enamel protein content is observed. Proteinases have traditionally been assumed to degrade the organic matrix prior to its removal from the enamel. Recently, a novel serine proteinase named enamel matrix serine proteinase-1 (EMSP1) was cloned from enamel organ epithelia. EMSP1 localizes primarily to the early maturation stage enamel and may, therefore, be involved in the degradation of proteins prior to their removal from the maturing enamel. Other, as yet unidentified, proteinases and proteinase inhibitors are almost certainly present within the forming enamel and await discovery.

Bartlett JD; Simmer JP

1999-01-01

163

Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus.  

UK PubMed Central (United Kingdom)

Asparaginyl proteinases (or legumains) are a recently identified, novel class of cysteine proteinase which specifically hydrolyse peptide bonds after asparagine residues. Legumains have been implicated in the activation of cysteine proteases, particularly cathepsin B-like proteinases which are thought to help degrade the bloodmeal in blood-feeding helminths such as schistosomes, hookworms and other nematode species. An EST sequence representing a full-length legumain was identified from the Haemonchus contortus dataset. This encoded a protein with a predicted Mr of 49 kDa, the amino acid sequence of which showed good homology (34-40% identity) to legumains from Schistosoma mansoni, human and rat and contained a legumain-like active site. RT-PCR indicated that the legumain transcript was expressed from the L4 life-cycle stage onwards. The coding sequence was expressed in E. coli and antibodies to the resultant recombinant protein indicated that the enzyme was expressed in the microvillar surface of the intestinal cells. Legumain activity was detected in extracts of the adult parasite but not the host protective Thiol-Sepharose-binding fraction, although it was detectable in the latter by immunoblot. Activity was relatively insensitive to E64, an inhibitor of cysteine proteinases and completely inhibited by the alkylating agent, N-ethylmaleimide, consistent with inhibitor effects on previously characterized legumains.

Oliver EM; Skuce PJ; McNair CM; Knox DP

2006-08-01

164

Evolutionary patterns of proteinase activity in attine ant fungus gardens  

DEFF Research Database (Denmark)

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.

Semenova, Tatyana; Hughes, David Peter

2011-01-01

165

Influence of air temperature on proteinase activity and beverage quality in Coffea arabica  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed (more) . Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

Abreu, Hellen Marília Couto de; Nobile, Paula Macedo; Shimizu, Milton Massao; Yamamoto, Paula Yuri; Silva, Emerson Alves; Colombo, Carlos Augusto; Mazzafera, Paulo

2012-01-01

166

Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals  

International Nuclear Information System (INIS)

Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward [3H]-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics

1989-01-01

167

Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals  

Energy Technology Data Exchange (ETDEWEB)

Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward ({sup 3}H)-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics.

Wu, Lan.

1989-01-01

168

Extracellular proteinase activity of Cryptococcus neoformans.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Extracellular proteinase activity was studied for eight strains of Cryptococcus neoformans var. neoformans and two strains of Cryptococcus neoformans var. gattii. Proteinase activity was measured by protein agar clearance, azoalbumin hydrolysis, gelatin liquefaction, and protein substrate polyacryla...

Chen, L C; Blank, E S; Casadevall, A

169

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases.  

Science.gov (United States)

Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera. PMID:15715970

Tamhane, Vaijayanti A; Chougule, Nanasaheb P; Giri, Ashok P; Dixit, Anirudha R; Sainani, Mohini N; Gupta, Vidya S

2005-01-12

170

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases.  

UK PubMed Central (United Kingdom)

Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.

Tamhane VA; Chougule NP; Giri AP; Dixit AR; Sainani MN; Gupta VS

2005-03-01

171

Proteinase trapping: screening for viral proteinase mutants by alpha complementation.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Many virally encoded proteinases cleave themselves out of a polyprotein, with cleavage occurring usually at their own N terminus. This property was used to develop an in vivo screening system using the lacZ gene fragment of M13mp18. When a fusion protein of the alpha fragment of beta-galactosidase a...

Liebig, H D; Skern, T; Luderer, M; Sommergruber, W; Blaas, D; Kuechler, E

172

Molecular Cloning and Characterization of Cystatin, a Cysteine Protease Inhibitor, from Bufo melanostictus.  

UK PubMed Central (United Kingdom)

Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.

Liu W; Ji S; Zhang AM; Han Q; Feng Y; Song Y

2013-10-01

173

Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.  

Science.gov (United States)

Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163. PMID:8419930

Kobayashi, M; Izui, H; Nagasawa, T; Yamada, H

1993-01-01

174

Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine endopeptidase (SH-EP).  

Science.gov (United States)

A cysteine endopeptidase, designated SH-EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH-EP is synthesized on membrane-bound ribosomes as an inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of the signal sequence; the 43-kDa intermediate of SH-EP is further processed to the 33-kDa mature enzyme via 39-kDa and 36-kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274-279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), were involved in post-translational processing of SH-EP in cotyledons of V. mungo seedlings. VmPE-1 was purified from the cotyledons as a protease that was involved in the processing of the 43-kDa intermediate to the 36-kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE-1 in the processing of SH-EP and related proteases in the cotyledons of germinated seeds. PMID:7635141

Okamoto, T; Minamikawa, T

1995-07-15

175

Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine endopeptidase (SH-EP).  

UK PubMed Central (United Kingdom)

A cysteine endopeptidase, designated SH-EP, occurs in the cotyledons of germinated seeds of Vigna mungo and acts to degrade the seed storage protein in protein storage vacuoles. SH-EP is synthesized on membrane-bound ribosomes as an inactive 45-kDa precursor, which is cotranslationally processed to a 43-kDa intermediate through cleavage of the signal sequence; the 43-kDa intermediate of SH-EP is further processed to the 33-kDa mature enzyme via 39-kDa and 36-kDa intermediates [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274-279]. The present in vitro processing experiments indicated that at least two processing enzymes, designated VmPE-1 and VmPE-2 (V. mungo processing enzymes 1 and 2), were involved in post-translational processing of SH-EP in cotyledons of V. mungo seedlings. VmPE-1 was purified from the cotyledons as a protease that was involved in the processing of the 43-kDa intermediate to the 36-kDa intermediate. The enzyme has a molecular mass of 33 kDa as estimated by SDS/polyacrylamide gel electrophoresis, and showed high similarity to the jackbean asparaginyl endopeptidase in terms of the primary structure and substrate specificity. We discuss the function of VmPE-1 in the processing of SH-EP and related proteases in the cotyledons of germinated seeds.

Okamoto T; Minamikawa T

1995-07-01

176

The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development  

Directory of Open Access Journals (Sweden)

Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia) facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin) system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

Barend Juan Vorster; Urte Schlüter; Magdeleen du Plessis; Stefan van Wyk; Matome Eugene Makgopa; Ignatious Ncube; Marian Dorcas Quain; Karl Kunert; Christine Helen Foyer

2013-01-01

177

Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections.  

UK PubMed Central (United Kingdom)

The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist.

El Sibaei MM; Abdel-Fattah NS; Ahmed SA; Abou-Seri HM

2012-04-01

178

Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections.  

Science.gov (United States)

The role of Trichomonas tenax as a pathogen had been clearly implicated in various pathological processes that arise outside the boundaries of the mouth. Although a relationship between the increased occurrence of this protozoan and progression of periodontal disease has been demonstrated, the ability of T. tenax in causing oral infections and the precise mechanism of tissue damage is not well known. The present study aimed to investigate different isolates of T.tenax from individuals having oral infections. Plaques and/or calculi samples were collected from 70 individuals who were diagnosed as having periodontitis and/or gingivitis, then subjected to parasitological examination and culture on modified trypticase, yeast and iron medium (TYI-S-33). Isolates successfully maintained in culture were further subjected to analysis of protein profile of lysates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and analysis of proteinases by non-denaturing gelatin-SDS-PAGE. Comparison of growth kinetics of seven T. tenax isolates showed a wide variability in the growth characteristics. Protein profiles of the seven isolates revealed a total 53 bands ranged in molecular weight (MW) from 5 to 95kDa using 12% resolution gel. Also, T. tenax isolates were found to possess 19 proteinase bands ranged in MW from 14 to 66kDa. The proteolytic bands were intensified by a cysteine proteinase activator and totally disappeared by treatment with a cysteine proteinase inhibitor suggesting that the proteinases were of cysteine proteinases type. The high frequency of T. tenax detected (28.6%) along with the variability in protein profiling and proteolytic activity of the isolates supports the possible pathogenicity of T. tenax and clarifies a conclusion that different strains with possibility of variable pathogenic potential may exist. PMID:22348932

El Sibaei, Mahmoud M; Abdel-Fattah, Nashwa S; Ahmed, Sabah A; Abou-Seri, Hanan M

2012-02-14

179

Proteinases of Trypanosoma cruzi: patential targets for the chemotherapy of Changas desease.  

Science.gov (United States)

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas Disease, contains cysteine, serine, threonine and metallo proteinases. Aspartic proteinases have not been found so far. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a complex mixture of isoforms by the major developmental stages of the parasite, including some membrane-bound isoforms. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus making the enzyme a very promising target for the development of new drugs against Chagas disease. In addition 30 kDa cathepsin B-like enzymes have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca(2+)-signalling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a serine carboxypeptidase belonging to the S10 family. Metalloproteinases homologous to the gp63 of Leishmania spp. are also present. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite. PMID:12171584

Cazzulo, Juan José

2002-11-01

180

Proteinases of Trypanosoma cruzi: patential targets for the chemotherapy of Changas desease.  

UK PubMed Central (United Kingdom)

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas Disease, contains cysteine, serine, threonine and metallo proteinases. Aspartic proteinases have not been found so far. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a complex mixture of isoforms by the major developmental stages of the parasite, including some membrane-bound isoforms. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus making the enzyme a very promising target for the development of new drugs against Chagas disease. In addition 30 kDa cathepsin B-like enzymes have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca(2+)-signalling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a serine carboxypeptidase belonging to the S10 family. Metalloproteinases homologous to the gp63 of Leishmania spp. are also present. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite.

Cazzulo JJ

2002-11-01

 
 
 
 
181

Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism.  

Science.gov (United States)

The mammalian liver tightly regulates its free cysteine pool, and intracellular cysteine in rat liver is maintained between 20 and 100 nmol/g even when sulfur amino acid intakes are deficient or excessive. By keeping cysteine levels within a narrow range and by regulating the synthesis of glutathione, which serves as a reservoir of cysteine, the liver addresses both the need to have adequate cysteine to support normal metabolism and the need to keep cysteine levels below the threshold of toxicity. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase (CDO) levels in the liver, with the turnover of CDO protein being dramatically decreased when intracellular cysteine levels increase. This occurs in response to changes in the intracellular cysteine concentration via changes in the rate of CDO ubiquitination and degradation. Glutathione synthesis also increases when intracellular cysteine levels increase as a result of increased saturation of glutamate-cysteine ligase (GCL) with cysteine, and this contributes to removal of excess cysteine. When cysteine levels drop, GCL activity increases, and the increased capacity for glutathione synthesis facilitates conservation of cysteine in the form of glutathione (although the absolute rate of glutathione synthesis still decreases because of the lack of substrate). This increase in GCL activity is dependent on up-regulation of expression of both the catalytic and modifier subunits of GCL, resulting in an increase in total catalytic subunit plus an increase in the catalytic efficiency of the enzyme. An important role of cysteine utilization for coenzyme A synthesis in maintaining cellular cysteine levels in some tissues, and a possible connection between the necessity of controlling cellular cysteine levels to regulate the rate of hydrogen sulfide production, have been suggested by recent literature and are areas that deserve further study. PMID:16702335

Stipanuk, Martha H; Dominy, John E; Lee, Jeong-In; Coloso, Relicardo M

2006-06-01

182

Emerging roles for proteinases in cancer.  

UK PubMed Central (United Kingdom)

Metalloproteinases and serine proteinases have been associated with tumor invasion and formation of metastasis which represent the major obstacles to cancer cure. The contribution of proteinases in these processes was initially thought to be the destruction of extracellular matrices. However, recent evidence suggests that they mainly affect tumor growth rather than invasion. Proteinases can indeed generate active matrix protein fragments, influence the release, the activation and the bioavailability of growth factors, and consequently modulate tumor cell growth, apoptosis and angiogenesis. Additionally, proteinases, their receptors and/or inhibitors can be directly involved in cell migration and in the processing or shedding of cell surface proteins. Further elucidation of the functions of proteinases is essential for the development of novel anticancer strategies.

Noël A; Gilles C; Bajou K; Devy L; Kebers F; Lewalle JM; Maquoi E; Munaut C; Remacle A; Foidart JM

1997-01-01

183

Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): Molecular characterization and transcriptional response upon immune stimulation.  

UK PubMed Central (United Kingdom)

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone.

Wickramaarachchi WD; De Zoysa M; Whang I; Wan Q; Lee J

2013-09-01

184

Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects  

Energy Technology Data Exchange (ETDEWEB)

Elevated levels of atmospheric carbon dioxide (CO{sub 2}), a consequence of anthropogenic global change, can profoundly affect the interactions between crop plants and insect pests and may promote yet another form of global change: the rapid establishment of invasive species. Elevated CO{sub 2} increased the susceptibility of soybean plants grown under field conditions to the invasive Japanese beetle (Popillia japonica) and to a variant of western corn rootworm (Diabrotica virgifera virgifera) resistant to crop rotation by down-regulating gene expression related to defense signaling [lipoxygenase 7 (lox7), lipoxygenase 8 (lox8), and 1-aminocyclopropane-1-carboxylate synthase (acc-s)]. The down-regulation of these genes, in turn, reduced the production of cysteine proteinase inhibitors (CystPIs), which are specific deterrents to coleopteran herbivores. Beetle herbivory increased CystPI activity to a greater degree in plants grown under ambient than under elevated CO{sub 2}. Gut cysteine proteinase activity was higher in beetles consuming foliage of soybeans grown under elevated CO{sub 2} than in beetles consuming soybeans grown in ambient CO{sub 2}, consistent with enhanced growth and development of these beetles on plants grown in elevated CO{sub 2}. These findings suggest that predicted increases in soybean productivity under projected elevated CO{sub 2} levels may be reduced by increased susceptibility to invasive crop pests.

Zavala, J.; Casteel, C.; DeLucia, E.; Berenbaum, M. [University of Illinois at Urbana-Champaign, Urbana, IL (United States)

2008-04-01

185

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase.  

UK PubMed Central (United Kingdom)

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.

Kristjansson MM; Kinsella JE

1990-08-01

186

Proteinase inhibitors in Brazilian leguminosae  

Directory of Open Access Journals (Sweden)

Full Text Available Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,000), Torresea cearensis (Mr = 13,000), Bauhinia pentandra (Mr = 20,000) and Bauhinia bauhinioides (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

C. A. M. Sampaio; M. L. V. Oliva; A. S. Tanaka; M. U. Sampaio

1991-01-01

187

Activation of snake venom metalloproteinases by a cysteine switch-like mechanism.  

UK PubMed Central (United Kingdom)

The cDNAs of several snake venom zinc endopeptidases code for a putative propeptide, which includes the conserved cysteine-containing sequence PKMCGVT. It has been suggested that binding of the cysteine thiol function to the active-site zinc, resulting in inactivation of the catalytic domain, occurs in a mode similar to the 'cysteine switch' mechanism proposed for matrix metalloproteinases. In order to confirm this hypothesis, inhibition kinetics have been performed on the metalloproteinase adamalysin II of the venom of the snake Crotalus adamanteus using several cysteine peptides. Among these the synthetic hexapeptide PKMCGV-NH2, corresponding to the conserved sequence portion of the known propeptides, was found to be by far the strongest inhibitor of this proteinase with a Ki of 3.4 microM. The inhibitory potencies of an equivalent peptide with the L-Cys replaced by a D-Cys or by an L-Ser as well as of reduced glutathione, cysteine and two unrelated cysteine peptides were by one to two orders of magnitudes lower. These findings strongly support a cysteine switch-like mechanism even for activation of the snake venom metalloproteinases.

Grams F; Huber R; Kress LF; Moroder L; Bode W

1993-11-01

188

Proteinases and their inhibitors in liver cancer  

Directory of Open Access Journals (Sweden)

Full Text Available Proteinases are known to be involved in many cancer-related processes, particularly in the breakdown of extracellular matrix barriers in the course of tumor invasion and metastasis. In this review we summarize the current knowledge about the role of the most important matrix-degrading proteinases (cathepsins, matrix metalloproteinases, plasmin/plasminogen activators) and their respective inhibitors in liver cancer progression and metastasis.

Verena Puxbaum, Lukas Mach

2009-01-01

189

Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori.  

UK PubMed Central (United Kingdom)

To identify proteinases involved in programmed cell death of the silk glands of Bombyx mori, we measured enzyme activities in silk gland homogenates. Several peptidyl-4-methylcoumaryl-7-amides (MCAs) and bovine hemoglobin were used as substrates in the presence and absence of proteinase inhibitors. The hydrolysis of t-butyloxycarbonyl-Phe-Ser-Arg-MCA (Boc-FSR-MCA), benzyloxy-carbonyl-Phe-Arg-MCA (Z-FR-MCA), and Z-Arg-Arg-MCA (Z-RR-MCA) was optimal at pH 5.5, 5.0, and 5.5, respectively. It was stimulated by the sulfhydryl compounds or EDTA and inhibited by both cysteine proteinase inhibitors and a cathepsin B-specific inhibitor, l-3-trans-(propyl-carbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-prolin (CA-074). The hemoglobin hydrolysis at the optimum pH 3.5 was inactivated by cysteine proteinase inhibitors, but stimulated slightly by pepstatin. The cleavage of Arg-MCA (R-MCA) and Leu-MCA (L-MCA) at optimum pH of 7.0 was strongly inhibited by an aminopeptidase inhibitor, puromycin, and by sulfhydryl compounds. The Boc-FSR-MCA, Z-FR-MCA, Z-RR-MCA, and hemoglobin hydrolyzing activities increased in the silk glands dramatically after cocoon formation, while the R-MCA and L-MCA cleaving activities declined. The results strongly suggest the involvement of cathepsin B- and cathepsin L-like proteinases in the histolysis of the silk gland during metamorphosis.

Shiba H; Uchida D; Kobayashi H; Natori M

2001-06-01

190

Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori.  

Science.gov (United States)

To identify proteinases involved in programmed cell death of the silk glands of Bombyx mori, we measured enzyme activities in silk gland homogenates. Several peptidyl-4-methylcoumaryl-7-amides (MCAs) and bovine hemoglobin were used as substrates in the presence and absence of proteinase inhibitors. The hydrolysis of t-butyloxycarbonyl-Phe-Ser-Arg-MCA (Boc-FSR-MCA), benzyloxy-carbonyl-Phe-Arg-MCA (Z-FR-MCA), and Z-Arg-Arg-MCA (Z-RR-MCA) was optimal at pH 5.5, 5.0, and 5.5, respectively. It was stimulated by the sulfhydryl compounds or EDTA and inhibited by both cysteine proteinase inhibitors and a cathepsin B-specific inhibitor, l-3-trans-(propyl-carbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-prolin (CA-074). The hemoglobin hydrolysis at the optimum pH 3.5 was inactivated by cysteine proteinase inhibitors, but stimulated slightly by pepstatin. The cleavage of Arg-MCA (R-MCA) and Leu-MCA (L-MCA) at optimum pH of 7.0 was strongly inhibited by an aminopeptidase inhibitor, puromycin, and by sulfhydryl compounds. The Boc-FSR-MCA, Z-FR-MCA, Z-RR-MCA, and hemoglobin hydrolyzing activities increased in the silk glands dramatically after cocoon formation, while the R-MCA and L-MCA cleaving activities declined. The results strongly suggest the involvement of cathepsin B- and cathepsin L-like proteinases in the histolysis of the silk gland during metamorphosis. PMID:11368511

Shiba, H; Uchida, D; Kobayashi, H; Natori, M

2001-06-01

191

Legumains and their functions in plants.  

UK PubMed Central (United Kingdom)

Legumains are a family of plant and animal Asn-specific cysteine proteinases with extra-cytoplasmic localization in vacuoles or cell walls. Plant legumains are involved in Asn-specific propolypeptide processing during, for example, storage-protein deposition in maturing seeds, when these proteins are resistant against degradation by legumains. With the transition to germination and subsequent seedling growth, storage proteins are opened to unlimited cleavage by legumains, which now contribute to protein mobilization. Here, we suggest a hypothesis that unifies both functions of legumains. Their action as propolypeptide-processing or protein-degrading enzymes is naturally controlled by the conformational state of their substrates, which undergo development- or environment-dependent changes. The suggested substrate conformation-dependent differential roles of legumains might not be restricted to seeds but could also apply to cells of different tissues in vegetative organs.

Müntz K; Shutov AD

2002-08-01

192

Isolation and identification of a new cysteine sulfoxide and volatile sulfur compounds from Allium subgenus Melanocrommyum.  

Science.gov (United States)

A new cysteine sulfoxide containing a pyridyl residue was identified in bulbs of Allium L. species belonging to the subgenus Melanocrommyum section Megaloprason. One of these species, Allium stipitatum, is widely used as a crop plant and in folk medicine in Central Asia. The new cysteine sulfoxide was identified as L-(+)-S-(2-pyridyl)-cysteine sulfoxide. Aside from this cysteine sulfoxide, several pyridyl compounds could be identified, which were formed out of cysteine sulfoxides by the action of alliinase. Found cysteine sulfoxides and their metabolites are chemically unstable; thus, the analysis is rather difficult. By combining high-performance liquid chromatography (HPLC), HPLC tandem mass spectrometry, NMR, IR, and photometric methods, full structure elucidation of the cysteine sulfoxide was possible. Alliinase reaction products were mainly determined by various MS techniques. The achieved results give new insights in the chemistry of Allium crop plants and are probably useful for chemotaxonomical classification of the subgenus Melanocrommyum. PMID:19919098

Kusterer, Jan; Vogt, Anja; Keusgen, Michael

2010-01-13

193

Isolation and identification of a new cysteine sulfoxide and volatile sulfur compounds from Allium subgenus Melanocrommyum.  

UK PubMed Central (United Kingdom)

A new cysteine sulfoxide containing a pyridyl residue was identified in bulbs of Allium L. species belonging to the subgenus Melanocrommyum section Megaloprason. One of these species, Allium stipitatum, is widely used as a crop plant and in folk medicine in Central Asia. The new cysteine sulfoxide was identified as L-(+)-S-(2-pyridyl)-cysteine sulfoxide. Aside from this cysteine sulfoxide, several pyridyl compounds could be identified, which were formed out of cysteine sulfoxides by the action of alliinase. Found cysteine sulfoxides and their metabolites are chemically unstable; thus, the analysis is rather difficult. By combining high-performance liquid chromatography (HPLC), HPLC tandem mass spectrometry, NMR, IR, and photometric methods, full structure elucidation of the cysteine sulfoxide was possible. Alliinase reaction products were mainly determined by various MS techniques. The achieved results give new insights in the chemistry of Allium crop plants and are probably useful for chemotaxonomical classification of the subgenus Melanocrommyum.

Kusterer J; Vogt A; Keusgen M

2010-01-01

194

A prodomain peptide of Plasmodium falciparum cysteine protease (falcipain-2) inhibits malaria parasite development.  

UK PubMed Central (United Kingdom)

Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite.

Korde R; Bhardwaj A; Singh R; Srivastava A; Chauhan VS; Bhatnagar RK; Malhotra P

2008-06-01

195

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strate (more) gy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Florent, I.; Le Bonniec, S.; Carcy, B.; Grellier, P.; Mercereau-Puijalon, O.; Bonnefoy, S.; Dhermy, D.; Monsigny, M.; Mayer, R.; Schrével, J.

1994-01-01

196

Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases.  

Science.gov (United States)

The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 A resolution and from SeMet-rGnk2 at 2.79 A resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2(1)3, with unit-cell parameters a = b = c = 143.2 A. PMID:17768341

Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi; Hatano, Ken-ichi; Tanokura, Masaru

2007-08-10

197

Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.  

Science.gov (United States)

Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by ?-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by ? -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively. PMID:23790889

Cerny, Christoph; Guntz-Dubini, Renée

2013-04-24

198

Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.  

UK PubMed Central (United Kingdom)

Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by ?-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by ? -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively.

Cerny C; Guntz-Dubini R

2013-11-01

199

Intranasal immunisation with a 62 kDa proteinase combined with cholera toxin or CpG adjuvant protects against Trichomonas vaginalis genital tract infections in mice.  

UK PubMed Central (United Kingdom)

Trichomonosis, caused by the protozoan parasite Trichomonas vaginalis, is one of the most frequent sexually transmitted diseases and is widely spread in all continents. Trichomonas vaginalis as well as other protozoan organisms have high levels of proteolitic activity mainly of the cysteine-proteinase type. This activity is necessary for recognition and adhesion of the parasite to the superficial epithelial cells of the host. In the present study, we show that intranasal immunisation with a 62 kDa cysteine-proteinase purified from T. vaginalis excretion-secretion products in combination with cholera toxin or with synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG-ODN) elicits 62kDa specific IgG and IgA in vaginal lavage fluid and specific IgG in serum. This immunisation protocol resulted in enhanced elimination of parasites following intravaginal challenge of BALB/c mice.

Hernández HM; Figueredo M; Garrido N; Sánchez L; Sarracent J

2005-11-01

200

Isolation and characterization of two forms of an acidic bromelain stem proteinase.  

UK PubMed Central (United Kingdom)

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

Harrach T; Eckert K; Maurer HR; Machleidt I; Machleidt W; Nuck R

1998-05-01

 
 
 
 
201

Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.  

UK PubMed Central (United Kingdom)

A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing.

Badgujar SB; Mahajan RT

2013-01-01

202

Peptide Mass Fingerprinting and N-Terminal Amino Acid Sequencing of Glycosylated Cysteine Protease of Euphorbia nivulia Buch.-Ham.  

Science.gov (United States)

A new cysteine protease named Nivulian-II has been purified from the latex of Euphorbia nivulia Buch.-Ham. The apparent molecular mass of Nivulian-II is 43670.846 Da (MALDI TOF/MS). Peptide mass fingerprint analysis revealed peptide matches to Maturase K (Q52ZV1_9MAGN) of Banksia quercifolia. The N-terminal sequence (DFPPNTCCCICC) showed partial homology with those of other cysteine proteinases of biological origin. This is the first paper to characterize a Nivulian-II of E. nivulia latex with respect to amino acid sequencing. PMID:23476742

Badgujar, Shamkant B; Mahajan, Raghunath T

2013-02-17

203

Conformational plasticity of the 2A proteinase from enterovirus 71.  

Science.gov (United States)

The 2A proteinase (2A(pro)) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A(pro) from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A(pro) was comprised of an N-terminal domain of a four-stranded antiparallel ? sheet and a C-terminal domain of a six-stranded antiparallel ? barrel with a tightly bound zinc atom. Unlike in other 2A(pro) structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A(pro) of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII ? strands. This was supported by molecular dynamic simulation. The structural variation among different 2A(pro) structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target. PMID:23616646

Cai, Qixu; Yameen, Muhammad; Liu, Weihua; Gao, Zhenting; Li, Yaozong; Peng, Xuanjia; Cai, Yaxian; Wu, Caiming; Zheng, Qian; Li, Jian; Lin, Tianwei

2013-04-24

204

Conformational plasticity of the 2A proteinase from enterovirus 71.  

UK PubMed Central (United Kingdom)

The 2A proteinase (2A(pro)) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A(pro) from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A(pro) was comprised of an N-terminal domain of a four-stranded antiparallel ? sheet and a C-terminal domain of a six-stranded antiparallel ? barrel with a tightly bound zinc atom. Unlike in other 2A(pro) structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A(pro) of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII ? strands. This was supported by molecular dynamic simulation. The structural variation among different 2A(pro) structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.

Cai Q; Yameen M; Liu W; Gao Z; Li Y; Peng X; Cai Y; Wu C; Zheng Q; Li J; Lin T

2013-07-01

205

A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.  

UK PubMed Central (United Kingdom)

Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. ?-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3?d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction.

Chae K; Gonong BJ; Kim SC; Kieslich CA; Morikis D; Balasubramanian S; Lord EM

2010-10-01

206

A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.  

Science.gov (United States)

Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. ?-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3?d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction. PMID:20667964

Chae, Keun; Gonong, Benedict J; Kim, Seung-Chul; Kieslich, Chris A; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M

2010-07-28

207

Proteinaceous cysteine protease inhibitors.  

UK PubMed Central (United Kingdom)

Studies of proteinaceous cysteine protease inhibitors originated with the discovery of cystatins in the 1960s. Since that time, a rich and fascinating world of proteins that control and regulate a multitude of important physiological processes, ranging from the basics of protein turnover to development and brain function, has been uncovered. Failures in such important and complex systems inevitably lead to pathologies. Many threatening diseases such as cancer or neurological disorders, to mention only some, are attributed to deregulation of protease-inhibitor balance. Moreover, important aspects of infection pathology and host defense rely on proteolysis and protease inhibition. Recent advances in the field of protease inhibitors have drawn attention to the possible use of this collected knowledge to control related pathological processes. This review attempts to familiarize the reader with proteinaceous cysteine protease inhibitors by providing an overview of current knowledge. The work primarily highlights biological processes in which the inhibitors are involved and focuses on pathologies resulting from aberrant protease-inhibitor balance, pointing out emerging possibilities for their correction.

Dubin G

2005-03-01

208

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft.  

UK PubMed Central (United Kingdom)

The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity.

Baker KC; Taylor MA; Cummings NJ; Tuñón MA; Worboys KA; Connerton IF

1996-06-01

209

Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft.  

Science.gov (United States)

The DNA coding for pro-papaya proteinase IV (PPIV) has been cloned and expressed in Escherichia coli. Heterologous expression of the protein, followed by refolding in vitro, yields an enzymatically active pro-enzyme which fails to autodigest to form the mature protein. Mutagenesis of the active site of papain to simulate that of PPIV yields a proenzyme which also fails to autoactivate. Complementary mutagenesis of the pro-region/mature boundary of PPIV, to introduce its own substrate recognition sequence, has, however, produced a pro-enzyme that will autocatalytically cleave. This is the first report of enzymatic activity in a recombinant pro-cysteine proteinase, and the first time that such a protein has been shown to fail to autocatalytically cleave because of its stringent substrate specificity. PMID:8862553

Baker, K C; Taylor, M A; Cummings, N J; Tuñón, M A; Worboys, K A; Connerton, I F

1996-06-01

210

Influence of culture medium pH and proteinase inhibitors on extracellular proteinase activity and cell growth of Sporothrix schenckii.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Sporothrix schenckii produces two extracellular proteinases in albumin- or collagen-supplemented unbuffered liquid medium. Proteinase I had an optimal pH of 6.0, and its activity was strongly inhibited by chymostatin. Proteinase II had an optimal pH of 3.5, and its activity was strongly inhibited by...

Tsuboi, R; Sanada, T; Ogawa, H

211

Two cysteine proteinases respond to bacterial and WSSV challenge in Chinese white shrimp Fenneropenaeus chinensis.  

Science.gov (United States)

The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time. PMID:20362060

Ren, Qian; Zhang, Xiao-Wen; Sun, Yun-Dong; Sun, Shan-Shan; Zhou, Jing; Wang, Zong-Heng; Zhao, Xiao-Fan; Wang, Jin-Xing

2010-03-31

212

Two cysteine proteinases respond to bacterial and WSSV challenge in Chinese white shrimp Fenneropenaeus chinensis.  

UK PubMed Central (United Kingdom)

The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12 h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2 h to 6 h and then recovered from 12 h to 24 h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time.

Ren Q; Zhang XW; Sun YD; Sun SS; Zhou J; Wang ZH; Zhao XF; Wang JX

2010-10-01

213

Channel catfish, Ictalurus punctatus, cysteine proteinases: cloning, characterisation and expression of cathepsin H and L.  

Science.gov (United States)

The antigen recognition by the host immune system is a complex biochemical process that requires a battery of enzymes. Cathepsins are one of the enzyme superfamilies involved in antigen degradation. We observed the up-regulation of catfish cathepsin H and L transcripts during the early stage of Edwardsiella ictaluri infection in preliminary studies, and speculated that cathepsin H and L may play roles in infection. We identified, sequenced and characterized the complete channel catfish cathepsin H and L cDNAs, which comprised 1415 and 1639 nucleotides, respectively. The open reading frames of cathepsin H appeared to encode a protein of 326-amino acid residues, which that of cathepsin L encoded a protein of 336 amino acids. The degree of conservation of the channel catfish cathepsin H and L amino acid sequences in comparison to other species ranged from 61% to 77%, and 67% to 85%, respectively. The catalytic triad and substrate binding sites are conserved in cathepsin H and L amino acid sequences. The cathepsin L transcript was expressed in all tissues examined, while the cathepsin H was expressed in restricted tissues. These results provide important information for further exploring the roles of channel catfish cathepsins in antigen processing. PMID:19084602

Yeh, Hung-Yueh; Klesius, Phillip H

2008-11-28

214

Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland  

International Nuclear Information System (INIS)

[en] Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5?. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 106. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His41, Asp86, Ser180; and six disulfide bridges Cys7-Cys139, Cys26-Cys42, Cys74-Cys232, Cys118-Cys186, Cys150-Cys165, Cys176-Cys201. Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 106, overtop the level of 105 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine proteinase gene exhibits strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. (authors)

2005-01-01

215

Expression, structure, and function of enamel proteinases.  

UK PubMed Central (United Kingdom)

Proteinases serve two important functions during dental enamel formation: They (a) process and (b) degrade enamel proteins. Different enzymes carry out these functions. Enamelysin (MMP-20) is the foremost enamel matrix-processing enzyme. Its expression initiates prior to the onset of dentin mineralization and continues throughout the secretory stage of amelogenesis. In vitro, enamelysin catalyzes all of the amelogenin cleavages that are known to occur during the secretory stage in vivo, and it is probably the enzyme responsible for the processing of all enamel proteins. There is evidence suggesting that enamelysin activity is critical for proper enamel formation. Uncleaved and processed enamel proteins often segregate into different compartments within the developing enamel layer, suggesting that they may have different functions. Intact ameloblastin and its C-terminal cleavage products localize in the superficial rod and interrod enamel, while its N-terminal cleavage products congregate in the sheath space. Intact enamelin is only present at the mineralization front within a micrometer of the enamel surface, while its cleavage products concentrate in the rod and interrod enamel. Processed enamel proteins accumulate during the secretory stage, but disappear early in the maturation stage. Enamel matrix serine proteinase 1 (EMSP1), now officially designated kallikrein 4 (KLK4), is believed to be the predominant degradative enzyme that clears enamel proteins from the matrix during maturation. KLK4 expression initiates during the transition stage and continues throughout maturation. KLK4 concentrates at the enamel surface when the enamel matrix disappears, and aggressively degrades amelogenin in vitro. During tooth development, proteinases are secreted by ameloblasts into the extracellular space, where they cleave enamel proteins by catalyzing the hydrolysis of peptide bonds. Enamel proteinases are present in low abundance and are not likely to participate directly in the mineralization process. Two major enamel proteinases have been identified: enamelysin (MMP20) and kallikrein 4 (KLK4). These proteinases are expressed at different times and have different functions. Their roles are to modify and/or to eliminate enamel matrix proteins, which affects the way enamel proteins interact with each other and with the developing enamel crystallites. A brief review of dental enamel formation is presented, followed by a more detailed analysis of enamelysin and KLK4 expression, structure, and function.

Simmer JP; Hu JC

2002-01-01

216

Cysteine proteases of malaria parasites.  

UK PubMed Central (United Kingdom)

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.

Rosenthal PJ

2004-12-01

217

Interactions of papaya proteinase IV with inhibitors.  

Science.gov (United States)

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues. PMID:1690669

Buttle, D J; Ritonja, A; Dando, P M; Abrahamson, M; Shaw, E N; Wikstrom, P; Turk, V; Barrett, A J

1990-03-12

218

Interactions of papaya proteinase IV with inhibitors.  

UK PubMed Central (United Kingdom)

Papaya proteinase IV (PPIV) is not inhibited by chicken cystatin, or human cystatins A or C, unlike most other proteinases of the papain superfamily. The enzyme inactivates chicken cystatin and human cystatin C by limited proteolysis of the glycyl bond previously shown to be involved in the inhibitory inactivity of the cystatins, but has no action on cystatin A. Contamination of commercial crystalline papain with PPIV accounts for the limited proteolysis of cystatins by 'papain' reported previously. PPIV is slowly bound by human alpha 2-macroglobulin. The enzyme is irreversibly inactivated by E-64, and by peptidyl diazomethanes containing glycine in P1 and a hydrophobic side-chain in P2. The reaction of PPIV with iodoacetate is extremely slow. PPIV is inhibited by peptide aldehydes despite the presence of bulky sidechains in P1, suggesting that these reversible inhibitors do not bind as substrate analogues.

Buttle DJ; Ritonja A; Dando PM; Abrahamson M; Shaw EN; Wikstrom P; Turk V; Barrett AJ

1990-03-01

219

Proteinases in bone resorption : obvious and less obvious roles  

DEFF Research Database (Denmark)

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Delaissé, Jean-Marie; Engsig, M T

2000-01-01

220

Specificity of a wheat gluten aspartic proteinase.  

UK PubMed Central (United Kingdom)

The substrate and peptide bond specificities of a purified wheat gluten aspartic proteinase (GlAP) are studied. GlAP shows maximum gluten hydrolysing activity at pH 3.0. At this pH, especially the wheat high molecular weight glutenin subunits (HMW-GS) and to a lesser extent the low molecular weight glutenin subunits and gliadins are hydrolysed. GlAP has no obvious effect on albumins and globulins. In its action on oxidised insulin B-chain, GlAP forms eight peptides and has high specificity for peptide bonds located between amino acid residues with large hydrophobic side chains (Leu, Phe, Tyr) but the peptide bond Glu13-Ala14 is also hydrolysed. Although structurally quite similar to a barley aspartic proteinase, the peptide bond specificity of GlAP towards oxidised insulin B-chain resembles slightly more that of a cardoon aspartic proteinase, cardosin B. HMW-GS 7, purified from cultivar Galahad-77, is rapidly hydrolysed by GlAP. N-Terminal amino acid sequence data show that GlAP cleaves at least one Met-Ile peptide bond at the end of the N-terminal domain and two Val-Leu peptide bonds in the repetitive domain of HMW-GS 7.

Bleukx W; Brijs K; Torrekens S; Van Leuven F; Delcour JA

1998-09-01

 
 
 
 
221

Specificity of a wheat gluten aspartic proteinase.  

Science.gov (United States)

The substrate and peptide bond specificities of a purified wheat gluten aspartic proteinase (GlAP) are studied. GlAP shows maximum gluten hydrolysing activity at pH 3.0. At this pH, especially the wheat high molecular weight glutenin subunits (HMW-GS) and to a lesser extent the low molecular weight glutenin subunits and gliadins are hydrolysed. GlAP has no obvious effect on albumins and globulins. In its action on oxidised insulin B-chain, GlAP forms eight peptides and has high specificity for peptide bonds located between amino acid residues with large hydrophobic side chains (Leu, Phe, Tyr) but the peptide bond Glu13-Ala14 is also hydrolysed. Although structurally quite similar to a barley aspartic proteinase, the peptide bond specificity of GlAP towards oxidised insulin B-chain resembles slightly more that of a cardoon aspartic proteinase, cardosin B. HMW-GS 7, purified from cultivar Galahad-77, is rapidly hydrolysed by GlAP. N-Terminal amino acid sequence data show that GlAP cleaves at least one Met-Ile peptide bond at the end of the N-terminal domain and two Val-Leu peptide bonds in the repetitive domain of HMW-GS 7. PMID:9748641

Bleukx, W; Brijs, K; Torrekens, S; Van Leuven, F; Delcour, J A

1998-09-01

222

CYSTEINE PROTEASES INHIBITORS  

UK PubMed Central (United Kingdom)

Substantially pure diastereoisomeric compounds of formula Ia, alternatively Ib, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, including hydrates, wherein PG is a protective group R 1 is a radical selected from the group consisting of phenylmethyl, 4-hydroxyphenylmethyl, (1 H -indol-3-yl)methyl and (1 H -imidazol-4-yl)methyl R 2 is a radical selected from the group consisting of -H, -CH 3 , -CH 2 SH, -CH 2 OH -CH 2 Ph, -CH 2 CO 2 H, -CH 2 CONH 2 , -CH(OH)CH 3 , -CH(CH 3 )CH 2 CH 3 , -CH(CH 3 ) 2 , -CH 2 CH(CH 3 ) 2 , -(CH 2 ) 2 SCH 3 , -(CH 2 ) 2 CO 2 H, -(CH 2 )CONH 2 , -(CH 2 ) 3 NHC(NH)NH 2 , -(CH 2 ) 4 NH 2 , imidazol-4-ylmethyl, 4-hydroxyphenylmethyl, (1 H -indol-3-yl)methyl, (1 H -imidazol-4-yl)methyl and -(CH 2 ) n -Ar and R 3 is a radical selected from the group consisting of -O(C 1 -C 4 )alkyl, -O(C 2 -C 4 )alkenyl, -O(C 2 -C 4 )alkynyl, -O(C 1 -C 4 )alkyl-Ar, -OAr, - NR a Ar, -N(R a )[(C 1 -C 4 )alkyl-Ar], and -NR a OAr and -N(R a )[O(C 1 -C 4 )alkyl-Ar]. These compounds are inhibitors of cysteine-proteases cruzain, rhodesain and falcipain, and therefore, useful in the treatment and/or prevention of pathologies such as Chagas disease, African trypanosomiasis or malaria.

GONZALEZ ADELANTADO FLORENCI VICENT; RODRIGUEZ PASTOR SANTIAGO; IZQUIERDO FERRER JAVIER

223

Cysteine-protease inhibitors  

UK PubMed Central (United Kingdom)

Substantially pure diastereoisomeric compounds of formula Ia, alternatively Ib, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, including hydrates, wherein PG is a protective group R1 is a radical selected from the group consisting of phenylmethyl, 4-hydroxyphenylmethyl, (1H-indol-3-yl)methyl and (1H-imidazol-4-yl)methyl R2 is a radical selected from the group consisting of -H, -CH3, -CH2SH, -CH2OH, -CH2Ph, -CH2CO2H, -CH2CONH2, -CH(OH)CH3, -CH(CH3)CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -(CH2)2SCH3, -(CH2)2CO2H, -(CH2)CONH2, -(CH2)3NHC(NH)NH2, -(CH2)4NH2, imidazol-4-ylmethyl, 4-hydroxyphenylmethyl, (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl and -(CH2)n-Ar and R3 is a radical selected from the group consisting of -O(C1-C4)alkyl, -O(C2-C4)alkenyl, -O(C2-C4)alkynyl, -O(C1-C4)alkyl-Ar, -OAr, -NRaAr, -N(Ra)[(C1-C4)alkyl-Ar], and -NRaOAr and -N(Ra)[O(C1-C4)alkyl-Ar]. These compounds are inhibitors of cysteine-proteases cruzain, rhodesain and falcipain, and therefore, useful in the treatment and/or prevention of pathologies such as Chagas disease, African trypanosomiasis or malaria.

GONZALEZ ADELANTADO FLORENCI VICENT; RODRIGUEZ PASTOR SANTIAGO; IZQUIERDO FERRER JAVIER

224

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family.  

UK PubMed Central (United Kingdom)

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.

Lombardi C; Ayach M; Beaurepaire L; Chenon M; Andreani J; Guerois R; Jupin I; Bressanelli S

2013-08-01

225

Proteinase inhibitors and activators strategic targets for therapeutic intervention.  

UK PubMed Central (United Kingdom)

The contributions in this meeting represent the state of the art in study of proteinases and their biological and therapeutic regulation. The near future of proteinase research will be fashioned by the new horizons of proteomics research [61], adding substance to the genomic data. Even new approaches in drug discovery, such as combinatorial chemistry, impact upon the understanding of the proteinase function, as with the discovery of a novel allosteric exosite in FVIIa by probing with libraries of ligands [62]. What is without question is that proteinases will remain at the forefront of understanding and intervention in human biochemistry and human disease pathology.

Deadman J

2000-09-01

226

Analysis of the solvent accessibility of cysteine residues on Maize rayado fino virus virus-like particles produced in Nicotiana benthamiana plants and cross-linking of peptides to VLPs.  

Science.gov (United States)

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials, vaccines, electronic materials, chemical tools, and molecular electronic containers. In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus, Carnation mottle virus, Cowpea chlorotic mottle virus, Tobacco mosaic virus, Turnip yellow mosaic virus, and MRFV. PMID:23439009

Natilla, Angela; Hammond, Rosemarie W

2013-02-14

227

Analysis of the solvent accessibility of cysteine residues on Maize rayado fino virus virus-like particles produced in Nicotiana benthamiana plants and cross-linking of peptides to VLPs.  

UK PubMed Central (United Kingdom)

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials, vaccines, electronic materials, chemical tools, and molecular electronic containers. In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus, Carnation mottle virus, Cowpea chlorotic mottle virus, Tobacco mosaic virus, Turnip yellow mosaic virus, and MRFV.

Natilla A; Hammond RW

2013-01-01

228

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD(®).  

UK PubMed Central (United Kingdom)

BACKGROUND AND OBJECTIVE: The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. MATERIAL AND METHODS: Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. RESULTS: Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. CONCLUSION: EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

Zilm PS; Bartold PM

2011-02-01

229

Molecular cloning, sequencing, expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity.  

UK PubMed Central (United Kingdom)

Cystatin, a superfamily of cysteine proteinase inhibitor of cathepsins and other cysteine proteinases, is widely distributed in animal tissues and body fluids. Although considerable attention has been given to mammalian and avian cystatins, little is known about cystatins from other vertebrates. In this study, a cDNA coding for Chinese sturgeon (Acipenser sinensis) cystatin was isolated and characterized. The corresponding mature cystatin peptide cDNA is 336 nucleotides long and encodes a protein of 112 amino acids. Sequence comparison showed that the cloned cystatin was a homolog of the mammalian Family II cystatin. The cystatin cDNA of Chinese sturgeon was subcloned into yeast expression vector pPICZalphaA and transformed into Pichia pastoris GS115 strain. After methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215 mg/l medium supernatant in shaking-flask fermentation medium, accounting for 73.6% of the total supernatant secreted proteins. Our data also showed that the recombinant cystatin is active in inhibiting the protease activity of papain and cathepsin B. Heat stability of the recombinant cystatin was also measured.

Bai J; Ma D; Lao H; Jian Q; Ye X; Luo J; Xong X; Li Y; Liang X

2006-09-01

230

Stress inducible proteinase inhibitor diversity in Capsicum annuum  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract 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.

Mishra Manasi; Mahajan Neha; Tamhane Vaijayanti A; Kulkarni Mahesh J; Baldwin Ian T; Gupta Vidya S; Giri Ashok P

2012-01-01

231

Modification of S1 subsite specificity in the cysteine protease cathepsin B.  

UK PubMed Central (United Kingdom)

Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases.

Fox T; Mason P; Storer AC; Mort JS

1995-01-01

232

Modification of S1 subsite specificity in the cysteine protease cathepsin B.  

Science.gov (United States)

Cysteine proteases of the papain family generally exhibit broad P1 specificity. A notable exception is papaya proteinase IV (PPIV), which only accepts Gly at this position. In all other cysteine proteases the S1 subsite residues 23 and 65 (papain numbering) are absolutely conserved as Gly, while in PPIV they are replaced by Glu and Arg, respectively. These differences appear to underlie both PPIV specificity and its resistance to inhibition by cystatins. To test this hypothesis, the equivalent residues (Gly27 and Gly73) in the mammalian cysteine protease cathepsin B were changed to Glu and Arg, respectively. Relative to the wild-type enzyme, the Gly27Glu and Gly73Arg mutants showed a drastic reduction in activity with substrates containing a P1 Arg. In contrast, substrates having a Gly residue in P1 were hydrolyzed effectively. The double mutant (Gly27Glu:Gly73Arg) exhibited no detectable activity against any substrate studied. Inhibition of the Gly73Arg mutant by E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane] was found to be similar to that of the wild-type enzyme. In contrast, inhibition by cystatin C exhibited a 20,000-fold reduction. These results demonstrate the dramatic influence of side chains at sequence locations 27 and 73 on the S1 subsite specificity of cysteine proteases. PMID:7770453

Fox, T; Mason, P; Storer, A C; Mort, J S

1995-01-01

233

PICORNAVIRUS L PROTEINASE AND METHODS OF MAKING AND USING THEREOF  

UK PubMed Central (United Kingdom)

Methods of making and using Picornavirus (foot-and-mouth disease virus) L proteinase peptides (PLPPs) are provided, including, but not limited to, expression of DNA encoding all or a portion thereof, such as a Picornavirus L proteinase (PLP) and variants thereof, as well as methods for determining active sites and inhibitors of a PLP.

SKERN Timothy; KIRCHWEGER Regina; ZIEGLER Elisabeth; BLAAS Dieter; LIEBIG Hans-Dieter; LAMPHEAR Barry; WATERS Debora; RHOADS Robert; SOMMERGRUBER Wolfgang; AHORN Horst

234

Picornavirus L proteinase and methods of making and using thereof  

UK PubMed Central (United Kingdom)

Methods of making and using Picornavirus L proteinase peptides (PLPPs) are provided, including, but not limited to, expression of DNA encoding all or a portion thereof, such as a Picornavirus L proteinase (PLP) and variants thereof, as well as methods for determining active sites and inhibitors of a PLP.

SKERN TIMOTHY; KIRCHWEGER REGINA; ZIEGLER ELISABETH; BLAAS DIETER; LIEBIG HANS-DIETER; LAMPHEAR BARRY J; WATERS DEBRA; RHOADS ROBERT E; SOMMERGRUBER WOLFGANG; AHORN HORST

235

[Suppression of activity of Candida albicans proteinases by cobalt chloride].  

UK PubMed Central (United Kingdom)

Influence of cobalt (II) chloride on the system of Candida albicans proteinase (SAP C. alb.) (both in solution and immobilized on a surface of nitrocellulose membranes) has been investigated. In solution cobalt chloride inactivated inducible but not constitute enzyme. In the heterogenous sytem proteolitical effect of the cobalt ion on inductible proteinase was also observed.

Kutyreva MP; Mukhametzianova AR; Ulakhovich NA

2012-07-01

236

Isolation and properties of extracellular proteinases from Sporothrix schenckii.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Sporothrix schenckii, mainly in the yeast form of the organism, produced extracellular proteinases when cultivated in liquid media containing albumin or collagen as a nitrogen source, but did not do so in brain heart infusion medium. Isolation of two extracellular proteinases from albumin-containing...

Tsuboi, R; Sanada, T; Takamori, K; Ogawa, H

237

Possible role of a proteinase in endosporulation of Coccidioides immitis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antis...

Yuan, L; Cole, G T; Sun, S H

238

First generation inhibitors of the adenovirus proteinase.  

UK PubMed Central (United Kingdom)

As there are more than 50 adenovirus serotypes, the likelihood of developing an effective vaccine is low. Here we describe inhibitors of the adenovirus proteinase (AVP) with the ultimate objective of developing anti-adenovirus agents. Inhibitors were identified via structure-based drug design using as druggable sites the active site and a conserved cofactor pocket in the crystal structures of AVP. A lead compound was identified that had an IC50 of 18 ?M. One of eight structural derivatives of the lead compound had an IC50 of 140 nM against AVP and an IC50 of 490 nM against the AVP with its cofactor bound.

McGrath WJ; Graziano V; Zabrocka K; Mangel WF

2013-08-01

239

Substrate specificity of proteinase yscA from saccharomyces cerevisiae.  

UK PubMed Central (United Kingdom)

Proteinase yscA is an intracellular aspartic proteinase located in the lysosome-like vacuole of the yeast cell. The specificity towards denatured protein substrates was determined by separation and identification of cleavage products after digestions with proteinase yscA, and compared to that obtained with pepsin used under similar conditions. Proteinase yscA is more selective towards the peptide bonds it cleaves than pepsin, but shows the same preference for large hydrophobic residues on both sides of the cleaved bond as pepsin and lysosomal cathepsin D. Phe, Leu and Glu are favoured in substrate subsite P1 and Phe, Ile, Leu and Ala in P'1, whereas Val is unfavoured in P'1. The implications for the role of proteinase yscA as hydrolase maturase are discussed.

Dreyer T

1989-01-01

240

The effect of proteinase inhibitors on glomerular albumin permeability induced in vitro by serum from patients with idiopathic focal segmental glomerulosclerosis.  

UK PubMed Central (United Kingdom)

BACKGROUND: The putative circulating factor responsible for the glomerular permeability alterations induced in vitro by serum from patients affected by focal segmental glomerulosclerosis (FSGS) remains unidentified. We have observed that a serine proteinase isolated from patient serum increases albumin permeability in isolated glomeruli. The objective of the present study was to determine the effect of various proteinase inhibitors on glomerular albumin permeability (P(alb)) in isolated glomeruli incubated with FSGS serum. METHODS: The study population consisted of 12 FSGS patients (eight males; mean age: 21+/-10 years) previously shown to have elevated serum albumin permeability activity. P(alb) was determined by measuring the change in glomerular volume induced by applying oncotic gradients to isolated healthy rat glomeruli treated with patient serum in comparison to control serum. Solutions of seven different proteinase inhibitors (0.5 mg/ml) were added to the incubation media with the sera (1:1 vol/vol): serine proteinase inhibitors (PMSF, leupeptin, aprotinin, gabexate mesylate), the cysteine proteinase inhibitor E-64, the metalloproteinase inhibitor EDTA and the aspartate proteinase inhibitor pepstatin. Sera from the same patients were also tested with the addition to the incubation media of quinaprilat, an inhibitor of the metalloproteinase angiotensin-converting enzyme. RESULTS: Mean P(alb) of the sera was 0.86+/-0.11, with the addition of PMSF 0.41+/-0.09, leupeptin 0.30+/-0.17, aprotinin 0.09+/-0.14, gabexate mesylate 0.27+/-0.25, E-64 0.81+/-0.09, EDTA 0.68+/-0.10 or pepstatin 0.76+/-0.11. The mean P(alb) of the sera combined with quinaprilat was reduced to 0.34+/-0.35. Thus, only the serine proteinase inhibitors consistently blocked the increased P(alb) induced by the FSGS sera. CONCLUSIONS: In the cascade of events that lead to the initiation of glomerular fibrosis in FSGS, the putative glomerular permeability factor associated with FSGS may require a serine proteinase to effect its activity.

Carraro M; Zennaro C; Artero M; Candiano G; Ghiggeri GM; Musante L; Sirch C; Bruschi M; Faccini L

2004-08-01

 
 
 
 
241

Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain?  

UK PubMed Central (United Kingdom)

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate and/or cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mg/g). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate-induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinate/g protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Lehmann A; Hagberg H; Orwar O; Sandberg M

1993-10-01

242

L-CYSTEINE PRODUCING MICROORGANISM AND METHOD FOR PRODUCING L-CYSTEINE  

UK PubMed Central (United Kingdom)

L-Cysteine is produced by culturing a microorganism having an ability to produce L-cysteine and modified so that expression of emrAB, emrKY, yojIH, acrEF, bcr, or cusA gene should be enhanced in a medium to produce and accumulate L-cysteine in the medium and collecting the L-cysteine from the medium. Genes coding for novel L-cysteine-excreting proteins are identified, and utilized for breeding of L-cysteine-producing microorganism to provide a novel method of producing L-cysteine.

TAKAGI HIROSHI; NAKAMORI SHIGERU; YAMAGUCHI AKIHITO; NISHINO KUNIHIKO

243

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.  

UK PubMed Central (United Kingdom)

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

Yang Y; Zhu YC; Ottea J; Husseneder C; Leonard BR; Abel C; Luttrell R; Huang F

2013-08-01

244

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.  

Science.gov (United States)

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes. PMID:23955944

Yang, Yunlong; Zhu, Yu Cheng; Ottea, James; Husseneder, Claudia; Leonard, B Rogers; Abel, Craig; Luttrell, Randall; Huang, Fangneng

2012-10-11

245

[Characterization of thermal denaturation process of proteinase K by spectrometry].  

UK PubMed Central (United Kingdom)

The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 degrees C, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4. 427 1 to 4. 032 4 ns and the fraction of alpha-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The alpha-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.

Zhang QB; Na XZ; Yin ZN

2013-07-01

246

Protease Inhibitors from Plants with Antimicrobial Activity  

Directory of Open Access Journals (Sweden)

Full Text Available Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

Jin-Young Kim; Seong-Cheol Park; Indeok Hwang; Hyeonsook Cheong; Jae-Woon Nah; Kyung-Soo Hahm; Yoonkyung Park

2009-01-01

247

Digestive proteinases from marine organisms and their applications  

Directory of Open Access Journals (Sweden)

Full Text Available Fish viscera have wide biotechnological potential as a source of digestive enzymes, especially proteinases. The biological diversity of fish species provides a wide array of enzymes with unique properties. Fish digestive proteolytic enzymes most commonly found include pepsin and trypsin. Those enzymes from fish viscera may have the advantages for the applications in the food industry since their temperature and other characteristics differ from homologous proteinases from warm-blooded animals. Therefore, digestive proteinases can be isolated as a value-added product from fish viscera and used as the processing aids in food industries to maximize the utilization of marine resources.

Sappasith Klomklao

2008-01-01

248

[Neutral proteinase in peroxisomes from rat liver cells  

UK PubMed Central (United Kingdom)

A proteinase with a pH optimum of 7.6 and Mr of 43 kD has been isolated from rat liver peroxisomes. The peroxisomes were shown to possess an intrinsic mechanism responsible for the degradation of proteins, e.g., catalase. Some physico-chemical properties and turnover parameters of neutral proteinase from peroxisomes (i.e., synthesis rate, lifetime, degradation of the newly synthesized enzyme) were studied. In terms of enzymatic and immunologic properties, the enzyme under study is similar to mitochondrial and cytosolic proteinases responsible for the initial inactivation of catalase in subcellular structures.

Komov VP; Strelkova MA; Makeeva AL

1988-09-01

249

Aspartic proteinases from Mucor spp. in cheese manufacturing.  

UK PubMed Central (United Kingdom)

Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing.

Yegin S; Fernandez-Lahore M; Jose Gama Salgado A; Guvenc U; Goksungur Y; Tari C

2011-02-01

250

Aspartic proteinases from Mucor spp. in cheese manufacturing.  

Science.gov (United States)

Filamentous fungi belonging to the order of Mucorales are well known as producers of aspartic proteinases depicting milk-clotting activity. The biosynthesis level, the biochemical characteristics, and the technological properties of the resulting proteinases are affected by the producer strain and the mode of cultivation. While the milk-clotting enzymes produced by the Rhizomucor spp. have been extensively studied in the past, much less is known on the properties and potential applications of the aspartic proteinases obtained for Mucor spp. Indeed, several Mucor spp. strains have been reported as a potential source of milk-clotting enzymes having unique technological properties. Both submerged fermentation and solid substrate cultivation are proven alternatives for the production of Mucor spp. aspartic proteinases. This review provides an overview on the bioprocessing routes to obtain large amounts of these enzymes, on their structural characteristics as related to their functional properties, and on their industrial applications with focus on cheese manufacturing. PMID:21127856

Yegin, Sirma; Fernandez-Lahore, Marcelo; Jose Gama Salgado, Antonio; Guvenc, Ulgar; Goksungur, Yekta; Tari, Canan

2010-12-03

251

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.  

UK PubMed Central (United Kingdom)

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Baskova IP; Zavalova LL

2001-07-01

252

Elafin is a potent inhibitor of proteinase 3.  

Science.gov (United States)

Elafin, a human skin derived inhibitor of human leukocyte elastase, was tested for inhibitory activity against proteinase 3, an elastin degrading proteinase of neutrophils. The inhibitory activity of elafin was compared with antileukoprotease and eglin C. Elafin proved to be a potent inhibitor of elastin-FITC degradation showing an IC 50 of 9.5 x 10(-9) M. Potency was found to be more than 100-fold higher as compared with antileukoprotease and eglin C. PMID:1989620

Wiedow, O; Lüademann, J; Utecht, B

1991-01-15

253

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

UK PubMed Central (United Kingdom)

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.

Rodrigues SP; Ventura JA; Aguilar C; Nakayasu ES; Choi H; Sobreira TJ; Nohara LL; Wermelinger LS; Almeida IC; Zingali RB; Fernandes PM

2012-06-01

254

Schistosoma japonicum, S. mansoni, S. haematobium, S. intercalatum, and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms.  

Science.gov (United States)

Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN2 only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species. PMID:9000231

Caffrey, C R; Rheinberg, C E; Moné, H; Jourdane, J; Li, Y L; Ruppel, A

1997-01-01

255

Schistosoma japonicum, S. mansoni, S. haematobium, S. intercalatum, and S. rodhaini: cysteine-class cathepsin activities in the vomitus of adult worms.  

UK PubMed Central (United Kingdom)

Vomitus from adults of five Schistosoma species was screened for biochemical homologues of the mammalian cysteine proteinases cathepsins B, H, and L. Bovine cathepsin B and rat cathepsin L served as references. Using the substrate Arg-NMec, a schistosome cathepsin H-like activity was never detected. All species degraded the cathepsin B substrate Z-Arg-Arg-NMec, but distinct species differences were observed with respect to pH optima and buffer preferences. The cathepsin B and L substrate Z-Phe-Arg-NMec was similarly degraded by all species, and activity was abolished by the cysteine proteinase inhibitor E-64. Preferences by vomitus proteinase activities for Z-Phe-Arg-NMec over Z-Arg-Arg-NMec were similar to or higher than those found for bovine cathepsin B but well below those observed for rat cathepsin L; also, the preferential cathepsin L inhibitor Z-Phe-PheCHN2 only partially inhibited proteinolytic activity. The results suggest the possible presence in vomitus of a minor cathepsin L-like activity and demonstrate a major cathepsin B-like activity that is biochemically variable between schistosome species.

Caffrey CR; Rheinberg CE; Moné H; Jourdane J; Li YL; Ruppel A

1997-01-01

256

Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis.  

UK PubMed Central (United Kingdom)

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.

Söllner C; Mentele R; Eckerskorn C; Fritz H; Sommerhoff CP

1994-02-01

257

Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis.  

Science.gov (United States)

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors. PMID:8112345

Söllner, C; Mentele, R; Eckerskorn, C; Fritz, H; Sommerhoff, C P

1994-02-01

258

[A serine proteinase from Thermoactinomyces vulgaris, strain INMI-4a  

UK PubMed Central (United Kingdom)

A serine proteinase was isolated from the cultural filtrate of the thermophylic actinomycet Thermoactinomycet vulgaris, strain INMI-4a. The purification procedure included affinity chromatography on bacitracin-Sepharose, ion-exchange separation on aminosilochrome, and gel-filtration on Sephadex G-25, resulting in a 194-fold purification and the 55% yield of the enzyme. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis in the presence of Na-SDS as well as by gel-filtration on Sephadex G-75 is equal to 28 000; the amino acid composition is: Lys11, His4, Arg5, Asp33, Thr22, Ser24, Glu16, Pro16, Gly30, Ala38, Cys1-2, Val20, Met1, Ile14, Leu8, Tyr16, The4, Trp6-7. The isoelectric point lies at pH 8--9; the pH optimum for the peptide substrate hydrolysis is Z-L-Ala-L-Ala-L-Leu-pNA is at 8.2. The enzyme is stable at pH 7--9. The temperature optimum of the proteolytic activity lies at 55 degrees; however, the enzyme is stable to heating for 1 h at 37 degrees. The proteinase is completely inactivated by the serine proteinase specific inhibitors--phenylmethylsulphofluoride and the protein inhibitor IT-AjT from Actinomyces, as well as by p-chloromercuribenzoate. The enzyme shows lytic activity against the cells of E. coli, Micrococcus lysodeicticus and of the yeasts. The Thermoactinomyces vulgaris serine proteinase, being definitely different from the serine proteinases from Actinomyces griseus, also reveals specific differences when compared to bacterial serine proteinases, e. g. subtilisins. There are some indications to the enzyme relationship with the family of carboxypeptidase Y-like serine proteinases.

Stepanov VM; Rudenskaia GN; Nesterova NG; Kupriianova TI; Khokhlova IuM

1980-10-01

259

[A serine proteinase from Thermoactinomyces vulgaris, strain INMI-4a].  

Science.gov (United States)

A serine proteinase was isolated from the cultural filtrate of the thermophylic actinomycet Thermoactinomycet vulgaris, strain INMI-4a. The purification procedure included affinity chromatography on bacitracin-Sepharose, ion-exchange separation on aminosilochrome, and gel-filtration on Sephadex G-25, resulting in a 194-fold purification and the 55% yield of the enzyme. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis in the presence of Na-SDS as well as by gel-filtration on Sephadex G-75 is equal to 28 000; the amino acid composition is: Lys11, His4, Arg5, Asp33, Thr22, Ser24, Glu16, Pro16, Gly30, Ala38, Cys1-2, Val20, Met1, Ile14, Leu8, Tyr16, The4, Trp6-7. The isoelectric point lies at pH 8--9; the pH optimum for the peptide substrate hydrolysis is Z-L-Ala-L-Ala-L-Leu-pNA is at 8.2. The enzyme is stable at pH 7--9. The temperature optimum of the proteolytic activity lies at 55 degrees; however, the enzyme is stable to heating for 1 h at 37 degrees. The proteinase is completely inactivated by the serine proteinase specific inhibitors--phenylmethylsulphofluoride and the protein inhibitor IT-AjT from Actinomyces, as well as by p-chloromercuribenzoate. The enzyme shows lytic activity against the cells of E. coli, Micrococcus lysodeicticus and of the yeasts. The Thermoactinomyces vulgaris serine proteinase, being definitely different from the serine proteinases from Actinomyces griseus, also reveals specific differences when compared to bacterial serine proteinases, e. g. subtilisins. There are some indications to the enzyme relationship with the family of carboxypeptidase Y-like serine proteinases. PMID:7016198

Stepanov, V M; Rudenskaia, G N; Nesterova, N G; Kupriianova, T I; Khokhlova, Iu M

1980-10-01

260

Proteinase activity and stability of natural bromelain preparations.  

UK PubMed Central (United Kingdom)

Bromelain is a complex mixture of proteinases typically derived from pineapple stem. Similar proteinases are also present in pineapple fruit. Beneficial therapeutic effects of bromelain have been suggested or proven in several human inflammatory diseases and animal models of inflammation, including arthritis and inflammatory bowel disease. However, it is not clear how each of the proteinases within bromelain contributes to its anti-inflammatory effects in vivo. Previous in vivo studies using bromelain have been limited by the lack of assays to control for potential differences in the composition and proteolytic activity of this naturally derived proteinase mixture. In this study, we present model substrate assays and assays for cleavage of bromelain-sensitive cell surface molecules can be used to assess the activity of constituent proteinases within bromelain without the need for biochemical separation of individual components. Commercially available chemical and nutraceutical preparations of bromelain contain predominately stem bromelain. In contrast, the proteinase activity of pineapple fruit reflects its composition of fruit bromelain>ananain approximately stem bromelain. Concentrated bromelain solutions (>50 mg/ml) are more resistant to spontaneous inactivation of their proteolytic activity than are dilute solutions, with the proteinase stability in the order of stem bromelain>fruit bromelain approximately ananain. The proteolytic activity of concentrated bromelain solutions remains relatively stable for at least 1 week at room temperature, with minimal inactivation by multiple freeze-thaw cycles or exposure to the digestive enzyme trypsin. The relative stability of concentrated versus dilute bromelain solutions to inactivation under physiologically relevant conditions suggests that delivery of bromelain as a concentrated bolus would be the preferred method to maximize its proteolytic activity in vivo.

Hale LP; Greer PK; Trinh CT; James CL

2005-04-01

 
 
 
 
261

Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.  

UK PubMed Central (United Kingdom)

Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes.

Taylor MA; Baker KC; Briggs GS; Connerton IF; Cummings NJ; Pratt KA; Revell DF; Freedman RB; Goodenough PW

1995-01-01

262

Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.  

Science.gov (United States)

Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes. PMID:7770454

Taylor, M A; Baker, K C; Briggs, G S; Connerton, I F; Cummings, N J; Pratt, K A; Revell, D F; Freedman, R B; Goodenough, P W

1995-01-01

263

Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases  

Energy Technology Data Exchange (ETDEWEB)

Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases being unreactive toward other classes of proteinases. Variation of the peptide portion of the reagents has provided affinity labels for the selective inactivation of the thiol endopeptidase cathepsin B, streptococcal proteinase, and clostripain and for the aminodipeptidase cathepsin C. Comparison of rates of inactivation of cathepsin B and streptococcal proteinase revealed a similarity in specificity, both enzymes showing a preference for a phenylalanine residue in the penultimate position of the peptide portion of the reagent. Reagents not satisfying the specificity of a particular thiol proteinase either did not inactivate or did so only very slowly. In some cases, the reagents bound to the enzyme in a manner unproductive for alkylation but productive for substrate behavior leading to destruction of the inhibitor by cleavage of the peptide portion. Thus, benzyloxycarbonyl-Phe-Gly-Phe diazomethyl ketone was rapidly cleaved by cathepsin B into benzyloxycarbonyl-Phe-Gly and phenylalanine diazomethyl ketone. Clostripain, an enzyme of trypsin-like specificity, was selectively inactivated by benzyloxycarbpnyl-Lys diazomethyl ketone at micromolar concentrations, but was inactivated extremely slowly by 2.5 x 10/sup -4/ M benzyloxycarbonyl-Phe-Ala diazomethyl ketone, a very effective inactivated by Gly-Phe diazomethyl ketone in the range of 10/sup -7/ to 10/sup -8/ M. By comparison, peptidyl diazomethyl ketones with blocked NH/sub 2/ terminals were considerably less effective. Peptidyl diazomethyl ketones are unreactive with such thiols as mercaptoethanol and glutathione.

Green, G.D.J.; Shaw, E.

1981-02-25

264

Inhibitors from pigeonpea active against lepidopteran gut proteinases.  

Science.gov (United States)

The proteinase inhibitors (PIs) active against bovine pancreatic trypsin, chymotrypsin, and insect midgut trypsin-like proteinases were found in the seeds of 14 cultivars and eight wild types of pigeonpea, Cajanus cajan L.. The inhibitory activity of PIs against trypsin and chymotrypsin, as well as their activity profile on gelatin-polyacrylamide gel electrophoresis (PAGE) were identical among the various cultivars. In contrast to cultivars, the wild types showed differences in inhibitory activity of PIs and their activity profile on gelatin-PAGE. The PIs from all cultivars and few wild types showed 10- to 50-fold higher activity against midgut trypsin-like proteinases of Achaea janata (L.) (Lepidoptera: Noctuidae), compared with bovine pancreatic trypsin. However, the PIs from both cultivars and wild types showed three- to nine-fold less activity against Spodoptera litura (F.) (Lepidoptera: Noctuidae) midgut trypsin-like proteinases, compared with bovine pancreatic trypsin. This inhibitory potential of PIs from cultivars and wild types, toward midgut trypsin-like proteinases from A. janata was further evident by the strong activity profile visualized on gelatin-PAGE. These results further suggest that the inhibitory potential of PIs from pigeonpea cultivars and wild types could be exploited in management of nonhost insects. PMID:20069866

Prasad, E R; Dutta-Gupta, A; Padmasree, K

2009-12-01

265

Inhibitors from pigeonpea active against lepidopteran gut proteinases.  

UK PubMed Central (United Kingdom)

The proteinase inhibitors (PIs) active against bovine pancreatic trypsin, chymotrypsin, and insect midgut trypsin-like proteinases were found in the seeds of 14 cultivars and eight wild types of pigeonpea, Cajanus cajan L.. The inhibitory activity of PIs against trypsin and chymotrypsin, as well as their activity profile on gelatin-polyacrylamide gel electrophoresis (PAGE) were identical among the various cultivars. In contrast to cultivars, the wild types showed differences in inhibitory activity of PIs and their activity profile on gelatin-PAGE. The PIs from all cultivars and few wild types showed 10- to 50-fold higher activity against midgut trypsin-like proteinases of Achaea janata (L.) (Lepidoptera: Noctuidae), compared with bovine pancreatic trypsin. However, the PIs from both cultivars and wild types showed three- to nine-fold less activity against Spodoptera litura (F.) (Lepidoptera: Noctuidae) midgut trypsin-like proteinases, compared with bovine pancreatic trypsin. This inhibitory potential of PIs from cultivars and wild types, toward midgut trypsin-like proteinases from A. janata was further evident by the strong activity profile visualized on gelatin-PAGE. These results further suggest that the inhibitory potential of PIs from pigeonpea cultivars and wild types could be exploited in management of nonhost insects.

Prasad ER; Dutta-Gupta A; Padmasree K

2009-12-01

266

Overexpression of a Weed (Solanum americanum) Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura  

Science.gov (United States)

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

Luo, Ming; Wang, Zhaoyu; Li, Huapeng; Xia, Kuai-Fei; Cai, Yinpeng; Xu, Zeng-Fu

2009-01-01

267

CYCLIC, CYSTEIN-FREE PROTEIN  

UK PubMed Central (United Kingdom)

A protein selected from the amino acid sequence of the region valine Val(91) to glycine Gly(l21) of the mature human tumour necrosis factor, or a portion thereof, with the proviso that the protein comprises at least the amino acid sequence of the region lysine Lys(98) to glutamic acid Glu(116), with the cysteine Cys(101) being replaced by a glycine and an amide bond being formed between the amino group of the side chain of the lysine Lys(98) and the carboxyl group of the side chain of the glutamic acid Glu(116), which activates epithelial ion channels and improves the lung function and which can be used for the manufacture of medicaments for the treatment of diseases associated with the lung function, such as oedemas.

FISCHER BERNHARD; LUCAS RUDOLF

268

The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast.  

UK PubMed Central (United Kingdom)

Peptidyl chloromethane and sulphonium salts containing multiple Arg and Lys residues were synthesized as potential inhibitors of prohormone and pro-protein processing proteinases. The potencies of these compounds were assayed by measuring the kinetics of inactivation of the yeast Kex2 proteinase, the prototype of a growing family of eukaryotic precursor processing proteinases. The most potent inhibitor, Pro-Nvl-Tyr-Lys-Arg-chloromethane, was based on cleavage sites in the natural Kex2 substrate pro-alpha-factor. This inhibitor exhibited a Ki of 3.7 nM and a second-order inactivation rate constant (k2/Ki) of 1.3 x 10(7) M-1.s-1 comparable with the value of kcat./Km obtained with Kex2 for the corresponding peptidyl methylcoumarinylamide substrate. The enzyme exhibited sensitivity to the other peptidyl chloromethanes over a range of concentrations, depending on peptide sequence and alpha-amino decanoylation, but was completely resistant to peptidyl sulphonium salts. Kinetics of inactivation by these new inhibitors of a set of 'control' proteinases, including members of both the trypsin and subtilisin families, underscored the apparent specificity of the compounds most active against Kex2 proteinase.

Angliker H; Wikstrom P; Shaw E; Brenner C; Fuller RS

1993-07-01

269

Effects of leupeptin on proteinase and germination of castor beans  

Energy Technology Data Exchange (ETDEWEB)

Leupeptin, tripeptide inhibitor of some proteinases, was shown previously to maintain the stability of several enzymes (isocitrate lyase, fumarase, and catalase) in crude extracts of castor bean endosperm. This reagent is now shown to inhibit the breakdown of water-soluble and crystalloid-storage proteins of the protein bodies isolated from castor beans by the SH-proteinase and it also inhibits the endopeptidase from mung beans. When suitably introduced into the endosperm of dry castor beans it strongly inhibits germination and seedling development. Application of leupeptin to endosperm halves removed from the seed prevents the normal development of enzymes concerned with gluconeogenesis from fat and drastically curtails sugar production. The results suggest that the SH-proteinase is intimately involved in the mobilization of storage proteins.

Alpi, A.; Beevers, H.

1981-10-01

270

Studies on the alkaline proteinase system in rat skeletal muscle.  

UK PubMed Central (United Kingdom)

The alkaline proteolytic activity, as measured with azocasein as substrate, is significantly increased in the musculus extensor digitorum longus 8 days after induction of diabetes mellitus, whereas in soleus muscle, a slightly lower activity is measured when compared to control rats. Ethyleneglycol extraction of the activity from muscles of normal rats and subsequent fractionation by gel filtration revealed three distinct proteinase activities, corresponding to mol. wts. of 120000, 70000 and 18000. Induction of diabetes mellitus in rats is followed by a loss of the 120000 mol.wt. component. Treatment of the diabetic rats with compound 48/80 results in a complete loss of all three proteinase peaks. The activity of a proteinase inhibitor with an apparent mol. wt. of 60000 was unaffected by these manipulations.

Dahlmann B; Kuehn L; Rutschmann M; Reinauer H

1981-01-01

271

Identification of the Murine Coronavirus MP1 Cleavage Site Recognized by Papain-Like Proteinase 2  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polypr...

Kanjanahaluethai, Amornrat; Jukneliene, Dalia; Baker, Susan C.

272

Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2.  

UK PubMed Central (United Kingdom)

Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.

Yamada F; Takahashi N; Murachi T

1976-06-01

273

Proteinase inhibitors from the medicinal leech Hirudo medicinalis.  

Science.gov (United States)

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities. PMID:11563948

Baskova, I P; Zavalova, L L

2001-07-01

274

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives.  

UK PubMed Central (United Kingdom)

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera.

Chougule NP; Hivrale VK; Chhabda PJ; Giri AP; Kachole MS

2003-10-01

275

Differential inhibition of Helicoverpa armigera gut proteinases by proteinase inhibitors of pigeonpea (Cajanus cajan) and its wild relatives.  

Science.gov (United States)

The seeds of 36 pigeonpea [Cajanus cajan (L) Millsp.] cultivars, resistant and susceptible to pests and pathogens and 17 of its wild relatives were analysed for inhibitors of trypsin, chymotrypsin, and insect gut proteinases to identify potential inhibitors of insect (Helicoverpa armigera) gut enzymes. Proteinase inhibitors (PIs) of pigeonpea cultivars showed total inhibition of trypsin and chymotrypsin, and moderate inhibition potential towards H. armigera proteinases (HGP). PIs of wild relatives exhibited stronger inhibition of HGP, which was up to 87% by Rhynchosia PIs. Electrophoretic detection of HGPI proteins and inhibition of HGP isoforms by few pigeonpea wild relative PIs supported our enzyme inhibitor assay results. Present results indicate that PIs exhibit wide range of genetic diversity in the wild relatives of pigeonpea whereas pigeonpea cultivars (resistant as well as susceptible to pests and pathogens) are homogeneous. The potent HGPIs identified in this study need further exploration for their use in strengthening pigeonpea defence against H. armigera. PMID:13679090

Chougule, Nanasaheb P; Hivrale, Vandana K; Chhabda, Pavanjeet J; Giri, Ashok P; Kachole, Manvendra S

2003-10-01

276

The structure of chagasin in complex with a cysteine protease clarifies the binding mode and evolution of an inhibitor family.  

UK PubMed Central (United Kingdom)

Protein inhibitors of proteolytic enzymes regulate proteolysis and prevent the pathological effects of excess endogenous or exogenous proteases. Cysteine proteases are a large family of enzymes found throughout the plant and animal kingdoms. Disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer, rheumatoid arthritis, osteoporosis, and emphysema. A family (I42) of cysteine protease inhibitors (http://merops.sanger.ac.uk) was discovered in protozoan parasites and recently found widely distributed in prokaryotes and eukaryotes. We report the 2.2 A crystal structure of the signature member of the I42 family, chagasin, in complex with a cysteine protease. Chagasin has a unique variant of the immunoglobulin fold with homology to human CD8alpha. Interactions of chagasin with a target protease are reminiscent of the cystatin family inhibitors. Protein inhibitors of cysteine proteases may have evolved more than once on nonhomologous scaffolds.

Wang SX; Pandey KC; Scharfstein J; Whisstock J; Huang RK; Jacobelli J; Fletterick RJ; Rosenthal PJ; Abrahamson M; Brinen LS; Rossi A; Sali A; McKerrow JH

2007-05-01

277

Partial immunological characterization of heat-stable proteinases from Pseudomonas spp. of dairy origin.  

UK PubMed Central (United Kingdom)

A homogeneous extracellular heat-stable proteinase from Pseudomonas fluorescens AH-70 was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relationship of the proteinases from 26 psychrotrophic Pseudomonas spp. isolated from raw milk. The inhibition of the proteinases by the antiserum, the gel precipitin reactions and the use of a double-antibody sandwich ELISA, revealed that proteinase AH-70 is immunologically related to proteinases from 8/26 other Pseudomonas strains. These results also indicate that the immunological techniques for the detection of proteolytic enzymes in raw milk require antibody preparations of broader specificity.

Azcona JI; Martín R; Hernández PE; Sanz B

1989-03-01

278

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

Science.gov (United States)

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. PMID:23117889

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

279

Proteinase K processing of rabbit muscle creatine kinase.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Udgivelsesdato: 1997-Jan , Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlate...

Leydier, C; Andersen, Jens S.; Couthon, F; Forest, E; Marcillat, O; Denoroy, L; Vial, C; Clottes, E

280

Proteinase 3 and prognosis of patients with acute myocardial infarction  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A multimarker approach may be useful for risk stratification in AMI (acute myocardial infarction) patients, particularly utilizing pathways that are pathophysiologically distinct. Our aim was to assess the prognostic value of PR3 (proteinase 3) in patients post-AMI. We compared the prognostic value ...

Ng, Leong L.; Khan, Sohail Q.; Narayan, Hafid; Quinn, Paulene; Squire, Iain B.; Davies, Joan E.

 
 
 
 
281

DIVERSITY OF DIGESTIVE PROTEINASES IN TENEBRIO MOLITOR (COLEOPTERA: TENEBRIONIDAE) LARVAE  

Science.gov (United States)

The spectrum of Tenebrio molitor larvae digestive proteinases was studied in the context of spatial organization of protein digestion in the midgut. The pH of midgut contents increased from 5.2–5.6 to 7.8–8.2 from the anterior to the posterior. This pH gradient was reflected in the pH optima of the ...

282

LARGE SCALE PURIFICATION OF PROTEINASES FROM CLOSTRIDIUM HISTOLYTICUM FILTRATES  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Conklin, David A. (Walter Reed Army Institute of Research, Washington, D. C.), Marion E. Webster, Patricia L. Altieri, Sanford Berman, Joseph P. Lowenthal, and Raymond B. Gochenour. Large scale purification of proteinases from Clostridium histolyticum filtrates. J. Bacteriol. 82:589–594. 1961.—A met...

Conklin, David A.; Webster, Marion E.; Altieri, Patricia L.; Berman, Sanford; Lowenthal, Joseph P.; Gochenour, Raymond B.

283

Isolation and characterization of an extracellular proteinase of Coccidioides immitis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A proteinase isolated from the respiratory pathogen, Coccidioides immitis, was shown to have collagenolytic and elastinolytic activity, as well as the ability to cleave human serum immunoglobulin G and secretory immunoglobulin A. Proteolytic activity was demonstrated with a bovine casein digestion a...

Yuan, L; Cole, G T

284

Proteinase inhibition by proform of eosinophil major basic protein (pro-MBP) is a multistep process of intra- and intermolecular disulfide rearrangements  

DEFF Research Database (Denmark)

The metzincin metalloproteinase pregnancy-associated plasma protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil major basic protein (pro-MBP), which forms a covalent 2:2 proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.

Pedersen, Simon Glerup; Boldt, Henning Bünsow

2005-01-01

285

Identification and characterization of bacterial cysteine dioxygenases: a new route of cysteine degradation for eubacteria.  

UK PubMed Central (United Kingdom)

In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic information about the active-site geometry of mammalian CDOs, we identified a total of four proteins from Bacillus subtilis, Bacillus cereus, and Streptomyces coelicolor A3(2) that shared low overall identity to CDO (13 to 21%) but nevertheless conserved important active-site residues. These four proteins were heterologously expressed and purified to homogeneity by a single-step immobilized metal affinity chromatography procedure. The ability of these proteins to oxidize cysteine to cysteine sulfinic acid was then compared against recombinant rat CDO. The kinetic data strongly indicate that these proteins are indeed bona fide CDOs. Phylogenetic analyses of putative bacterial CDO homologs also indicate that CDO is distributed among species within the phyla of Actinobacteria, Firmicutes, and Proteobacteria. Collectively, these data suggest that a large subset of eubacteria is capable of cysteine sulfoxidation. Suggestions are made for how this novel pathway of cysteine metabolism may play a role in the life cycle of the eubacteria that have it.

Dominy JE Jr; Simmons CR; Karplus PA; Gehring AM; Stipanuk MH

2006-08-01

286

Purification and characterization of multiple forms of the pineapple-stem-derived cysteine proteinases ananain and comosain.  

UK PubMed Central (United Kingdom)

A mixture of ananain (EC 3.4.22.31) and comosain purified from crude pineapple stem extract was found to contain numerous closely related enzyme forms. Chromatographic separation of the major enzyme forms was achieved after treatment of the mixture with thiol-modifying reagents: reversible modification with 2-hydroxyethyl disulphide provided enzyme for kinetic studies, and irreversible alkylation with bromotrifluoroacetone or iodoacetamide gave enzyme for structural analyses by 19F-n.m.r. and electrospray mass spectrometry respectively. Structural and kinetic analyses revealed comosain to be closely related to stem bromelain (EC 3.4.22.32), whereas ananain differed markedly from both comosain and stem bromelain. Nevertheless, differences were seen between comosain and stem bromelain in amino acid composition and kinetic specificity towards the epoxide inhibitor E-64. Differences between five isolatable alternative forms of ananain were characterized by amidolytic activity, thiol stoichiometry and accurate mass determinations. Three of the enzyme forms displayed ananain-like amidolytic activity, whereas the other two forms were inactive. Thiol-stoichiometry determinations revealed that the active enzyme forms contained one free thiol, whereas the inactive forms lacked the reactive thiol required for enzyme activity. M.s. provided direct evidence for oxidation of the active-site thiol to the corresponding sulphinic acid.

Napper AD; Bennett SP; Borowski M; Holdridge MB; Leonard MJ; Rogers EE; Duan Y; Laursen RA; Reinhold B; Shames SL

1994-08-01

287

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)  

Directory of Open Access Journals (Sweden)

Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

M.E. Pereira; F.A. Dörr; N.C. Peixoto; J.F. Lima-Garcia; F. Dörr; G.G. Brito

2005-01-01

288

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae).  

UK PubMed Central (United Kingdom)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4 degrees C and the supernatants were used in enzymatic assays at 30 degrees C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 +/- 0.15 absorbance variation min(-1) mg protein(-1), at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 +/- 0.02 mmol p-nitroaniline min(-1) mg protein(-1). The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50 degrees C, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Pereira ME; Dörr FA; Peixoto NC; Lima-Garcia JF; Dörr F; Brito GG

2005-11-01

289

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was (more) 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Pereira, M.E.; Dörr, F.A.; Peixoto, N.C.; Lima-Garcia, J.F.; Dörr, F.; Brito, G.G.

2005-11-01

290

Neutrophil elastase and proteinase-3 trigger G-protein biased signaling through proteinase activated receptor-1 (PAR1).  

UK PubMed Central (United Kingdom)

Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Since PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesised that neutrophil- derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase (NE) and proteinase-3 (PR3) cleave the human PAR1 N-terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands (TLs) unmasked by NE and PR3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated Mitogen Activated Protein Kinase (MAPK) activation. This signaling was blocked by pertussis toxin, implicating a G?i triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical TL revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity.

Mihara K; Ramachandran R; Renaux B; Saifeddine M; Hollenberg MD

2013-09-01

291

Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo.  

Science.gov (United States)

The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. coli cells by the expression of cDNA on pCEN1. beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. coli carrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coli culture. beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E. coli carrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants. PMID:8280125

Noji, M; Murakoshi, I; Saito, K

1993-12-30

292

Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo.  

UK PubMed Central (United Kingdom)

The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. coli cells by the expression of cDNA on pCEN1. beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. coli carrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coli culture. beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E. coli carrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.

Noji M; Murakoshi I; Saito K

1993-12-01

293

The aspartic proteinase family of three Phytophthora species  

Science.gov (United States)

Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs) are produced in a wide variety of species (from bacteria to humans) and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively) were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the A? peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during their translocation into the host red blood cell. Since the translocation of Phytophthora effector proteins is currently a topic of intense research activity, the identification in Phytophthora of potential functional homologues of plasmepsin V would appear worthy of investigation. Indeed, elucidation of the physiological roles of the APs identified here offers areas for future study. The significant revision of gene models and detailed annotation presented here should significantly facilitate experimental design.

2011-01-01

294

The aspartic proteinase family of three Phytophthora species  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs) are produced in a wide variety of species (from bacteria to humans) and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively) were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the A? peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during their translocation into the host red blood cell. Since the translocation of Phytophthora effector proteins is currently a topic of intense research activity, the identification in Phytophthora of potential functional homologues of plasmepsin V would appear worthy of investigation. Indeed, elucidation of the physiological roles of the APs identified here offers areas for future study. The significant revision of gene models and detailed annotation presented here should significantly facilitate experimental design.

Kay John; Meijer Harold JG; ten Have Arjen; van Kan Jan AL

2011-01-01

295

Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase  

Energy Technology Data Exchange (ETDEWEB)

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with /sup 51/Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of /sup 51/Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H/sub 2/O/sub 2/; (iii) cytolysis of H/sub 2/O/sub 2/-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. The authors conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase.

Pontremoli, S.; Melloni, E.; Michetti, M.; Sacco, O.; Sparatore, B.; Salamino, F.; Damiani, G.; Horecker, B.L.

1986-03-01

296

Astrocytes and the regulation of cerebral cysteine/cystine redox potential: implications for cysteine neurotoxicity  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The sulfur amino acid, cysteine plays an essential role in maintaining cellular redox potential and is a key constituent of the antioxidant, glutathione. Cysteine is highly reactive and readily oxidises to the disulfide form, cystine, producing oxygen radicals as a by-product. Extracellular oxidisin...

McBean, Gethin J.

297

Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli.  

Science.gov (United States)

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells. PMID:12883870

Awano, N; Wada, M; Kohdoh, A; Oikawa, T; Takagi, H; Nakamori, S

2003-03-20

298

Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli.  

UK PubMed Central (United Kingdom)

In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts. Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC. It was also found that TNase synthesis was induced by the presence of L-cysteine. The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid. These results indicated that TNase and CBL did act on L-cysteine degradation in E. coli cells.

Awano N; Wada M; Kohdoh A; Oikawa T; Takagi H; Nakamori S

2003-08-01

299

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

UK PubMed Central (United Kingdom)

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.

Beton D; Guzzo CR; Ribeiro AF; Farah CS; Terra WR

2012-09-01

300

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

Science.gov (United States)

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. PMID:22659439

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

2012-05-30

 
 
 
 
301

Dispersal of Bap-mediated Staphylococcus aureus biofilm by proteinase K.  

UK PubMed Central (United Kingdom)

The dominant role of biofilm-associated protein (Bap) in Staphylococcus aureus biofilm development prompted us to investigate Bap as a potential target for proteinase-mediated biofilm dispersion. Biofilm assay in microtitre plates showed that proteinase K hampered the early adhesion of cells as well as biofilm development. Proteinase K treatment of 24- and 48-h-old biofilms showed enhanced dispersion of bap-positive S. aureus biofilm; however, proteinase K did not affect the bap-negative S. aureus biofilm. When antibiotics were used in combination with proteinase K, significant enhancement in antibiotic action was noticed against bap-positive S. aureus biofilm. This study establishes that antibiotics in combination with proteinase K can be used for controlling S. aureus biofilms in whose development Bap surface protein has a major role. We propose that Bap protein could be a potential target for therapeutic control of S. aureus infections (for example, bovine mastitis).

Kumar Shukla S; Rao TS

2013-02-01

302

Production of a heterologous proteinase A by Saccharomyces kluyveri  

DEFF Research Database (Denmark)

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.

MØller, K; Tidemand, L D

2001-01-01

303

Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates.  

UK PubMed Central (United Kingdom)

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.

Bartlett JD

2013-01-01

304

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.  

UK PubMed Central (United Kingdom)

Abstract One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Odei-Addo F; Frost C; Smith N; Ogawa T; Muramoto K; Oliva ML; Gráf L; Naude R

2013-10-01

305

Benzoquinone reveals a cysteine-dependent desensitization mechanism of TRPA1.  

UK PubMed Central (United Kingdom)

The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel.

Ibarra Y; Blair NT

2013-05-01

306

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain.  

UK PubMed Central (United Kingdom)

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease.

Øverbye A; Sætre F; Hagen LK; Johansen HT; Seglen PO

2011-09-01

307

Autophagic activity measured in whole rat hepatocytes as the accumulation of a novel BHMT fragment (p10), generated in amphisomes by the asparaginyl proteinase, legumain.  

Science.gov (United States)

To investigate the stepwise autophagic-lysosomal processing of hepatocellular proteins, the abundant cytosolic enzyme, betaine:homocysteine methyltransferase (BHMT) was used as a probe. Full-length (45 kDa) endogenous BHMT was found to be cleaved in an autophagy-dependent (3-methyladenine-sensitive) manner in isolated rat hepatocytes to generate a novel N-terminal 10-kDa fragment (p10) identified and characterized by mass spectrometry. The cleavage site was consistent with cleavage by the asparaginyl proteinase, legumain and indeed a specific inhibitor of this enzyme (AJN-230) was able to completely suppress p10 formation in intact cells, causing instead accumulation of a 42-kDa intermediate. To prevent further degradation of p10 or p42 by the cysteine proteinases present in autophagic vacuoles, the proteinase inhibitor leupeptin had to be present. Asparagine, an inhibitor of amphisome-lysosome fusion, did not detectably impede either p42 or p10 formation, indicating that BHMT processing primarily takes place in amphisomes rather than in lysosomes. Lactate dehydrogenase (LDH) was similarly degraded primarily in amphisomes by leupeptin-sensitive proteolysis, but some additional leupeptin-resistant LDH degradation in lysosomes was also indicated. The autophagic sequestration of BHMT appeared to be nonselective, as the accumulation of p10 (in the presence of leupeptin) or of its precursors (in the additional presence of AJN-230) proceeded at approximately the same rate as the model autophagic cargo, LDH. The complete lack of a cytosolic background makes p10 suitable for use in a "fragment assay" of autophagic activity in whole cells. Incubation of hepatocytes with ammonium chloride, which neutralizes amphisomes as well as lysosomes, caused rapid, irreversible inhibition of legumain activity and stopped all p10 formation. The availability of several methods for selective targeting of legumain in intact cells may facilitate functional studies of this enigmatic enzyme, and perhaps suggest novel ways to reduce its contribution to cancer cell metastasis or autoimmune disease. PMID:21610319

Øverbye, Anders; Sætre, Frank; Hagen, Linda Korseberg; Johansen, Harald Thidemann; Seglen, Per O

2011-09-01

308

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi.  

UK PubMed Central (United Kingdom)

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas' heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

Monteiro AC; Abrahamson M; Lima AP; Vannier-Santos MA; Scharfstein J

2001-11-01

309

Sulfide Production from Cysteine by Desulfovibrio desulfuricans  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two rumen nitrate-reducing isolates of Desulfovibrio desulfuricans were found to hydrolyze cysteine with the production of sulfide and pyruvate. When cultured on agar medium containing yeast extract with nitrate as the primary electron acceptor and ferrous chloride as the indicator, blackening of co...

Forsberg, C. W.

310

Determining cysteine oxidation status using differential alkylation  

Science.gov (United States)

Oxidative damage to proteins plays a major role in aging and in the pathology of many degenerative diseases. Under conditions of oxidative stress, reactive oxygen and nitrogen species can modify key redox sensitive amino acid side chains leading to altered biological activities or structures of the targeted proteins. This in turn can affect signaling or regulatory control pathways as well as protein turnover and degradation efficiency in the proteasome. Cysteine residues are particularly susceptible to oxidation, primarily through reversible modifications (e.g., thiolation and nitrosylation), although irreversible oxidation can lead to products that cannot be repaired in vivo such as sulfonic acid. This report describes a strategy to determine the overall level of reversible cysteine oxidation using a stable isotope differential alkylation approach in combination with mass spectrometric analysis. This method employs 13C-labeled alkylating reagents, such as N-ethyl-[1,4-13C2]-maleimide, bromo-[1,2-13C2]-acetic acid and their non-labeled counterparts to quantitatively assess the level of cysteine oxidation at specific sites in oxidized proteins. The differential alkylation protocol was evaluated using standard peptides and proteins, and then applied to monitor and determine the level of oxidative damage induced by diamide, a mild oxidant. The formation and mass spectrometric analysis of irreversible cysteine acid modification will also be discussed as several such modifications have been identified in subunits of the mitochondrial electron transport chain complexes. This strategy will hopefully contribute to our understanding of the role that cysteine oxidation plays in such chronic diseases such as Parkinson's disease, where studies in animal and cell models have shown oxidative damage to mitochondrial Complex I to be a specific and early target.

Schilling, Birgit; Yoo, Chris B.; Collins, Christopher J.; Gibson, Bradford W.

2004-08-01

311

Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.  

UK PubMed Central (United Kingdom)

RNA silencing limits transient expression of heterologous proteins in plants. Co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. Little is known whether silencing suppression could be improved without harm to plant tissues. This study reports development of enhanced silencing suppressors by engineering the helper component proteinase (HCpro) of Potato virus A (PVA). Mutations were introduced to a short region of HCpro (positions 330-335 in PVA HCpro), which is hypervariable among potyviruses. Three out of the four HCpro mutants suppressed RNA silencing more efficiently and sustained expression of co-expressed jellyfish green fluorescent protein for a longer time than wild-type HCpro in agroinfiltrated leaves of Nicotiana benthamiana. Leaf tissues remained healthy-looking without any visible signs of stress.

Haikonen T; Rajamäki ML; Valkonen JP

2013-11-01

312

Mapping von Willebrand factor A domain binding sites on a snake venom metalloproteinase cysteine-rich domain.  

Science.gov (United States)

The PIII class of the snake venom metalloproteinases (SVMPS) are acknowledged to be one of the major hemorrhage producing toxins in crotalid venoms. This class of SVMPS are structurally distinguished by the presence of disintegrin-like and cysteine-rich domains carboxy to the metalloproteinase domain and thus share structural homology with many of the ADAMs proteins. It has been suggested that the presence of the carboxy domain are the key structural determinants for potent hemorrhagic activity in that they may serve to target the proteinases to specific key extracellular matrix and cell surface substrates for proteolysis leading to hemorrhage production at the capillaries. Following from previous studies in our laboratory in this investigation we scanned the cysteine-rich domain of the PIII hemorrhagic SVMP jararhagin using synthetic peptides in an attempt to identify regions which could bind to von Willebrand factor (vWF), a known binding partner for jararhagin. From these studies we identified two such peptide, Jar6 and Jar7 that could support binding to vWF as well as block the recombinant cysteine-rich domain of jararhagin binding to vWF. Using the coordinates for the recently solved crystal structure of the PIII SVMP VAP1, we modeled the structure of jararhagin and attempted to dock the modeled cysteine-rich structure of that protein to the A1 domain of vWF. These studies indicated that effective protein-protein interaction between the two ligands was possible and supported the data indicating that the Jar6 peptide was involved, whereas the Jar7 peptide was observed to be sterically blocked from interaction. In summary, our studies have identified a region on the cysteine-rich domain of a PIII SVMP that interacts with vWF and based on molecular modeling could be involving in the interaction of the cysteine-rich domain of the SVMP with the A1 domain of vWF thus serving to target the toxin to the protein for subsequent proteolytic degradation. PMID:17118332

Pinto, Antonio F M; Terra, Renata M S; Guimaraes, Jorge A; Fox, Jay W

2006-11-10

313

Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata.  

UK PubMed Central (United Kingdom)

Research into the molecular basis of plant-insect interactions is hampered by the inability to alter the expression of individual genes in plants growing under natural conditions. The ability of virus-induced gene silencing (VIGS) to silence the expression of two jasmonate-induced genes known to mediate the expression of two potent direct defences (nicotine and proteinase inhibitors) that are produced in different tissues (roots and shoots, respectively) in Nicotiana attenuata is documented here. Fragments of consensus sequences of N. attenuata's putrescine N-methyltransferase (PMT) and trypsin inhibitor (TI) genes were cloned in sense, anti-sense and inverted repeat orientations into the Tobacco Rattle Virus (TRV) to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in plants previously elicited with methyl jasmonate (MeJA) or left as controls. MeJA treatment elicited 2.4- and 9.8-fold increases in the concentrations of nicotine and proteinase inhibitors, respectively, and inoculation with constructs containing appropriate genes inhibited these MeJA-induced increases and halved constitutive accumulations, regardless of the orientation of the gene fragment. Root PMT transcript levels were significantly elevated in MeJA-treated plants 10 h after elicitation, but not in plants inoculated with the appropriate TRV constructs 9 d prior to MeJA treatment, demonstrating that VIGS was responsible for the inhibition of these potent direct defences. While additional research is required to minimize the effects on plant growth and the risks of using such constructs in natural settings, it is concluded that VIGS has a potential to manipulate the expression of genes important for ecological interactions.

Saedler R; Baldwin IT

2004-01-01

314

A novel proteinase, SCO4, is required for photosynthetic acclimation to higher light intensities in Arabidopsis.  

UK PubMed Central (United Kingdom)

Excess light can have a negative impact on photosynthesis, thus plants have evolved many different ways to adapt to different light conditions to both optimize energy use and avoid damage caused by excess light. Analysis of the snowy cotyledon 4 (sco4) mutant revealed a mutation in a chloroplast-targeted protein which shares limited homology with CaaX-type-endopeptidases. The SCO4 protein possesses an important function in photosynthesis and development, with point mutations rendering the seedlings and adult plants susceptible to photo-oxidative stress. The sco4 mutation impairs acclimation of chloroplasts and their photosystems to excess light, evidenced in a reduction in PS I function, decreased linear electron transfer, yet increased non-photochemical quenching. SCO4 is localized to the chloroplasts and suggests the existence of an unreported type of protein modification within this organelle. Phylogenetic and yeast complementation analyses of SCO4-like proteins reveals that SCO4 is a member of a unknown group of higher plant-specific proteinases quite distinct from the well described CaaX-type endopeptidases RCE1 and STE24 and lacks canonical CaaX activity. Therefore, we hypothesize that SCO4 is a novel endopeptidase required for critical protein modifications within chloroplasts, influencing the function of proteins involved in photosynthesis required for tolerance to excess light.

Albrecht V; Kauss D; Fan D; Hu Y; Collinge D; Marri S; Chow FW; Liebers M; Pfannschmidt T; Apel K; Pogson BJ

2013-08-01

315

Isolation of a keratinolytic proteinase from Trichophyton mentagrophytes with enzymatic activity at acidic pH.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A keratinolytic proteinase with enzyme activity at acidic pH was isolated from culture filtrates of Trichophyton mentagrophytes, a major pathogenic fungus of dermatophytosis. The molecular weight of the proteinase was estimated to be 41,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresi...

Tsuboi, R; Ko, I; Takamori, K; Ogawa, H

316

A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 re...

Welch, A R; Woods, A S; McNally, L M; Cotter, R J; Gibson, W

317

Coexpression of Aspartic Proteinases and Human Leukocyte Antigen-DR in Human Transplanted Lung  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Aspartic proteinases have recently been shown to be implicated in antigen processing. We explored the expression of two aspartic proteinases, cathepsins E and D, and of human leukocyte antigen-DR (HLA-DR) molecules in a consecutive series of 80 transbronchial biopsies from transplanted lungs. For co...

Arbustini, Eloisa; Morbini, Patrizia; Diegoli, Marta; Grasso, Maurizia; Fasani, Roberta; Vitulo, Patrizio; Fiocca, Roberto

318

Cloning, expression and characterization of the proteinase from human herpesvirus 6.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation ...

Tigue, N J; Matharu, P J; Roberts, N A; Mills, J S; Kay, J; Jupp, R

319

Production, Characterization, and Epitope Mapping of a Monoclonal Antibody against Aspartic Proteinase of Candida albicans  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigat...

Na, Byoung-Kuk; Chung, Gyung-Tae; Song, Chul-Yong

320

Isolation and expression of a gene which encodes a wall-associated proteinase of Coccidioides immitis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

A chymotrypsinlike serine proteinase of Coccidioides immitis with an estimated molecular size of 34 kDa has been shown by immunoelectron microscopy to be associated with the walls of the parasitic cells of this human respiratory pathogen. The proteinase has been suggested to play a role in spherule ...

Cole, G T; Zhu, S W; Hsu, L L; Kruse, D; Seshan, K R; Wang, F

 
 
 
 
321

Cloning of the bovine leukemia virus proteinase in Escherichia coli and comparison of its specificity to that of human T-cell leukemia virus proteinase.  

UK PubMed Central (United Kingdom)

The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein. The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies. The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration. Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1. Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced.

Zahuczky G; Boross P; Bagossi P; Emri G; Copeland TD; Oroszlan S; Louis JM; Tözsér J

2000-03-01

322

Cloning of the bovine leukemia virus proteinase in Escherichia coli and comparison of its specificity to that of human T-cell leukemia virus proteinase.  

Science.gov (United States)

The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein. The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies. The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration. Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1. Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced. PMID:10719169

Zahuczky, G; Boross, P; Bagossi, P; Emri, G; Copeland, T D; Oroszlan, S; Louis, J M; Tözsér, J

2000-03-16

323

Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor.  

UK PubMed Central (United Kingdom)

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.

Lomas DA; Evans DL; Upton C; McFadden G; Carrell RW

1993-01-01

324

In praise of impurity: 30S ribosomal S15 protein-assisted crystallization of turnip yellow mosaic virus proteinase.  

Science.gov (United States)

Turnip yellow mosaic virus is an excellent model for eukaryotic positive-stranded RNA virus replication. Correct processing of the replication polyprotein is dependent on the virally encoded cysteine proteinase (PRO) domain. Crystalline needles obtained from highly pure preparations of the recombinant 17.6 kDa PRO did not diffract. In contrast, small hexagonal prisms that were obtained together with the needles under the same conditions but from a poorly purified preparation diffracted to 2 Å resolution and allowed structure determination by MIRAS. It turned out that the hexagonal crystals contained stoichiometric amounts of PRO and Escherichia coli 30S ribosomal S15, a 10.1 kDa protein commonly co-purified by immobilized metal-affinity chromatography. The solvent content is nearly 70%, with S15 bridging parallel infinite helices of PRO across large solvent channels. With hindsight, this spurious interaction not only yielded diffraction-quality crystals but would also have allowed structure determination by molecular replacement using S15 as a search model and subsequent automatic rebuilding of the asymmetric unit. PMID:22505427

Robin, Charlotte; Beaurepaire, Lionel; Chenon, Mélanie; Jupin, Isabelle; Bressanelli, Stéphane

2012-03-28

325

In praise of impurity: 30S ribosomal S15 protein-assisted crystallization of turnip yellow mosaic virus proteinase.  

UK PubMed Central (United Kingdom)

Turnip yellow mosaic virus is an excellent model for eukaryotic positive-stranded RNA virus replication. Correct processing of the replication polyprotein is dependent on the virally encoded cysteine proteinase (PRO) domain. Crystalline needles obtained from highly pure preparations of the recombinant 17.6 kDa PRO did not diffract. In contrast, small hexagonal prisms that were obtained together with the needles under the same conditions but from a poorly purified preparation diffracted to 2 Å resolution and allowed structure determination by MIRAS. It turned out that the hexagonal crystals contained stoichiometric amounts of PRO and Escherichia coli 30S ribosomal S15, a 10.1 kDa protein commonly co-purified by immobilized metal-affinity chromatography. The solvent content is nearly 70%, with S15 bridging parallel infinite helices of PRO across large solvent channels. With hindsight, this spurious interaction not only yielded diffraction-quality crystals but would also have allowed structure determination by molecular replacement using S15 as a search model and subsequent automatic rebuilding of the asymmetric unit.

Robin C; Beaurepaire L; Chenon M; Jupin I; Bressanelli S

2012-04-01

326

The long road of research on snake venom serine proteinases.  

UK PubMed Central (United Kingdom)

It has long been recognized that snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, blood pressure and platelet aggregation. Therefore, SVSPs have been used as refined tools to study molecular mechanisms involved in the activation of key factors that control hemostasis and as therapeutic agents in various thrombotic and hemostatic conditions. The aim of this review is to highlight the state of our knowledge on the advances made in SVSP research since the 18th century. It includes the personal accounts of some distinguished scientists that addressed specific problems and contributed to advance the field.

Serrano SM

2013-02-01

327

Proteinase-mediated insect resistance to Bacillus thuringiensis toxins.  

UK PubMed Central (United Kingdom)

Two Bacillus thuringiensis (Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs.

Oppert B; Kramer KJ; Beeman RW; Johnson D; McGaughey WH

1997-09-01

328

Cleavage specificity of coxsackievirus 3C proteinase for peptide substrate.  

UK PubMed Central (United Kingdom)

The substrate requirements of coxsackievirus 3C proteinase (3Cpro) were investigated on the C-terminal side of the scissile bond using C-terminal truncated peptides of the substrate peptide Ac-EALFQGPPV. Not only the Gln-Gly bond of Ac-EALFQG-NH2 but also the C-terminal amide group of Ac-EALFQ-NH2 was hydrolyzed by 3Cpro, suggesting that the essential residues for cleavage by coxsackievirus 3Cpro would exist within the N-terminal 5 residues.

Miyashita K; Okunishi J; Utsumi R; Komano T; Tamura T; Satoh N

1996-04-01

329

Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida).  

UK PubMed Central (United Kingdom)

Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman-Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus.

Simões RA; Silva-Filho MC; Moura DS; Delalibera I Jr

2008-03-01

330

Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida).  

Science.gov (United States)

Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman-Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus. PMID:18357504

Simões, R A; Silva-Filho, M C; Moura, D S; Delalibera, I

2008-03-21

331

Dermatophyte Trichophyton mentagrophytes Produces Cysteine Protease Inhibitor.  

UK PubMed Central (United Kingdom)

The protein inhibitor of cysteine proteases was isolated from an important zoophilic dermatophyte species Trichophyton mentagrophytes (T. mentagrophytes) and partially characterized. The isolation process involved affinity chromatography, followed by ion-exchange chromatography and reverse phase high performance liquid chromatography. The fungal inhibitor appears to exist in a high (24 kDa) and low (12 kDa) molecular mass form. It inhibits proteolytic activity of papain, cathepsins B and L but not of cathepsin H or trypsin. Results of immunoblotting procedures indicate that sera of T. mentagrophytes infected rabbits contain antibodies against higher molecular mass forms of the inhibitor. Since no sequence homology has been found between partial protein sequences of T. mentagrophytes inhibitor and other known cysteine protease inhibitors so far, we can speculate that this inhibitor has some structurally unique characteristics. The T. mentagrophytes inhibitor shares some biochemical similarities (molecular mass, high and low molecular mass forms, inhibitory profiles) with clitocypin from Clitocybe nebularis and macrocypins from Macrolepiota procera.

Premrov-Bajuk B; Zdovc I; Smrekar V; Križaj I; Leonardi A; Drobni?-Košorok M

2011-03-01

332

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole.  

Science.gov (United States)

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed. PMID:11022031

Okamoto, T; Toyooka, K; Minamikawa, T

2001-01-01

333

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole.  

UK PubMed Central (United Kingdom)

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.

Okamoto T; Toyooka K; Minamikawa T

2001-01-01

334

Cysteine peptidase inhibitors in trypanosomatid parasites.  

UK PubMed Central (United Kingdom)

Parasitic diseases caused by pathogenic protozoa remain a challenge for public health. Despite efforts to control transmission, to improve early diagnosis and to optimize patient care, millions of infected people, mainly in poor areas of the globe, develop debilitating pathologies that are often fatal. For most of those disorders, the current treatments are greatly unsatisfactory and the continuous search for alternative chemotherapies remains at the center of research. Over the last decades, cysteine peptidases of protozoa feature as highly promising drug targets and their validation in laboratory models of disease or experimental infections instigated growing efforts in medicinal chemistry, aiming at the development of compounds with therapeutical potential. More recently, it was uncovered that protozoa also express new families of endogenous proteinaceous peptidase inhibitors that act as potential virulence factors. In this review, we will cover the main findings that contributed to the validation of cysteine peptidases from Trypanosoma cruzi, Trypanosoma brucei and Leishmania as drug targets and the current knowledge of their biological roles in those organisms. We give an overview of the development of small molecule cysteine peptidase inhibitors with anti-parasite activity and describe the current background on natural peptidase inhibitors in trypanosomatids.

Lima AP; Reis FC; Costa TF

2013-01-01

335

The role of inflammation and proteinases in tumor progression.  

UK PubMed Central (United Kingdom)

Chronic inflammation is an important risk factor for the development of cancers. The link between chronic inflammation and the risk of developing cancer is now well established. At least 20% of all cancers arise in association with infection and chronic inflammation. Inflammation and cancer are linked both along intrinsic (driven by genetic events causing malignancy) and extrinsic (driven by inflammatory conditions predisposing to tumor) pathways. Proteinases are key contributors to the breakdown and reconstitution of extracellular matrix components in physiological processes and pathological conditions, including destructive diseases and tumor progression. Matrix metalloproteinases are especially essential in the complex process of coregulation between cellular components of the tumor environment, and they are considered as potential diagnostic and prognostic biomarkers in many types and stages of cancer. Although the link between chronic inflammation, proteinases and risk of developing cancer is now well established, several open questions remain. The most exciting challenge is to find the best approach to target cancer-associated inflammation in patients with cancer. With respect to matrix metalloproteinases, the development of a new generation of selective inhibitors is a promising area of research.

Herszényi L; Lakatos G; Hritz I; Varga MZ; Cierny G; Tulassay Z

2012-01-01

336

Diisopropyl fluorophosphate labeling of sperm-associated proteinases  

International Nuclear Information System (INIS)

[en] Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm

1990-01-01

337

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida/ Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese INTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produção de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e (more) proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3%) dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4%) foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7%). Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2%). Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (?2=1.9 p=0.5901 (?2=1.9 p=0.5901). CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas. Abstract in english INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical i (more) solates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p

D'Eça Júnior, Aurean; Silva, Anderson França; Rosa, Fernanda Costa; Monteiro, Sílvio Gomes; Figueiredo, Patrícia de Maria Silva; Monteiro, Cristina de Andrade

2011-06-01

338

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida  

Directory of Open Access Journals (Sweden)

Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (pINTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produção de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3%) dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4%) foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7%). Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2%). Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (?2=1.9 p=0.5901 (?2=1.9 p=0.5901). CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas.

Aurean D'Eça Júnior; Anderson França Silva; Fernanda Costa Rosa; Sílvio Gomes Monteiro; Patrícia de Maria Silva Figueiredo; Cristina de Andrade Monteiro

2011-01-01

339

Specific cysteine protease inhibitors act as deterrents of western flower thrips, Frankliniella occidentalis (Pergande), in transgenic potato.  

Science.gov (United States)

In this study, the effects of the accumulation of cysteine protease inhibitors on the food preferences of adult female western flower thrips, Frankliniella occidentalis (Pergande), were investigated. Representative members of the cystatin and thyropin gene families (stefin A, cystatin C, kininogen domain 3 and equistatin) were expressed in potato (Solanum tuberosum) cv. Impala, Kondor and Line V plants. In choice assays, a strong time- and concentration-dependent deterrence from plants expressing stefin A and equistatin was observed. Cystatin C and kininogen domain 3 were not found to be active. All tested inhibitors were equally or more active than stefin A at inhibiting the proteolytic activity of thrips, but, in contrast with stefin A, they were all expressed in potato as partially degraded proteins. The resistance of cysteine protease inhibitors against degradation in planta by endogenous plant proteases may therefore be relevant in explaining the observed differences in the deterrence of thrips. The results demonstrate that, when given a choice, western flower thrips will select plants with low levels of certain cysteine protease inhibitors. The novel implications of the defensive role of plant cysteine protease inhibitors as both deterrents and antimetabolic proteins are discussed. PMID:17168890

Outchkourov, Nikolay S; de Kogel, Willem Jan; Schuurman-de Bruin, Antje; Abrahamson, Magnus; Jongsma, Maarten A

2004-09-01

340

Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.  

Science.gov (United States)

Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci. PMID:20621193

Donpudsa, Suchao; Söderhäll, Irene; Rimphanitchayakit, Vichien; Cerenius, Lage; Tassanakajon, Anchalee; Söderhäll, Kenneth

2010-07-16

 
 
 
 
341

Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P(2) position in proteinase inhibitory activity.  

UK PubMed Central (United Kingdom)

Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hemocytes and contain an uncommon P(2) amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P(1) Ser and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P(2) amino acid residue from Gly to Pro, mimicking the P(2) residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P(2) residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci.

Donpudsa S; Söderhäll I; Rimphanitchayakit V; Cerenius L; Tassanakajon A; Söderhäll K

2010-11-01

342

Transamination of L-cysteine sulfinate in the growing rat.  

Directory of Open Access Journals (Sweden)

Full Text Available The enzyme activities involved in the transamination of L-cysteine sulfinate (L-alanine 3-sulfinic acid), L-aspartate and L-cysteine were examined in fetal, neonatal and maternal rat liver and placenta. In fetal and neonatal rat liver, aminotransferase activity was most active with L-cysteine sulfinate as a substrate and was also active with L-aspartate, while activity with L-cysteine was very low. The activity of transamination of L-cysteine sulfinate in rat liver developed in parallel with that of L-aspartate and L-cysteine. The aminotransferase activity markedly increased after the 19th day of gestation, reaching the same value as adult liver on the 3rd day after birth. The ratios of transamination of L-cysteine sulfinate to that of L-aspartate and to that of L-cysteine were constant during development. These observations suggest that L-cysteine sulfinate, L-aspartate and L-cysteine are transaminated by the same enzyme in the rat liver during development. Since placental aminotransferase activity was extremely low compared with that of the liver, it was suggested that the placenta did not play an important role in the transamination of these amino acids during pregnancy.

Akahori,Shuichiro; Ejiri,Kohei; Kanemori,Hirofumi; Kudo,Takafumi; Sekiba,Kaoru; Ubuka,Toshihiko; Akagi,Reiko

1987-01-01

343

Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane  

International Nuclear Information System (INIS)

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki=3.3 nM) and baupain (Ki=2.1x10-8 M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125 nM), cathepsin K (Ki=0.76 nM), cathepsin L (Ki=0.6 nM), and cathepsin V (Ki=1.0 nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.

2004-08-06

344

Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains  

Directory of Open Access Journals (Sweden)

Full Text Available Direct extraction of DNA from natural environment and clinical samples has become a useful alternative for the phylogenetic identification and in situ detection of individual microbial cells without cultivation. In this study, three different Gram positive microorganisms (B. cereus, B. subtilis, and S. aureus) were chosen for genomic DNA extraction. High salt SDS (Sodium Dodesyl Sulfate) based extraction method was followed to extract genomic DNA with addition of three different lysis protocols to observe the effect of proteinase-K on total genomic DNA yield, lysis steps were carried with SDS, SDS with 3 µl proteinase-K and SDS with 6µl proteinase-K. High molecular weight intact DNA bands were observed only for Bacillus subtilis when the extraction procedure was carried out in presence of SDS, SDS with proteinase-K (3µl) and SDS with increased amount of proteinase-K (6µl). In presence of SDS and increased amount of proteinase-K (6µl) the mean value of DNA concentration for Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus were found to be 1.53±0.15, 1.36±0.10 and 1.65±0.10 µg/µl respectively. However, in absence of proteinase-K, the mean values of DNA concentration were found to be decreased (1.28±0.10, 1.34±0.15, 1.23±0.10 µg/µl for B. cereus, B. subtilis, and S. aureus respectively) for all these stains. Although in case of B. subtilis the overall effect of proteinase-K was not found to be significant in terms of DNA concentration and DNA band intensity, however, for B. cereus, and S. aureus sharp decrease in total extracted DNA concentration was observed suggesting the increased lysis effect of proteinase-K on the thick peptidoglycan layer of Gram-positive cell wall such as B. cereus, and S. aureus.

Mohammad Shahriar; Md. Rashidul Haque; Shaila Kabir; Irin Dewan; Mohiuddin Ahmed Bhuyian

2011-01-01

345

Novel insights into the delayed vasospasm following subarachnoid haemorrhage: importance of proteinase signalling.  

UK PubMed Central (United Kingdom)

This article summarizes the findings of Kameda et al. (this issue of BJP) that suggest a new avenue for the pharmacological treatment of subarachnoid haemorrhage (SAH) involving the combined use of a proteinase inhibitor (argatroban) that targets thrombin and an antioxidant (vitamin C). The findings are presented in the context of previous modalities of treating SAH that are of modest impact and the possibility that inhibiting proteinase-mediated signalling via proteinase-activated receptors like the thrombin PAR1 receptor combined with blocking oxidative stress may provide a new avenue for the treatment of SAH.

Hollenberg MD

2012-01-01

346

Orphan PTMs: Rare, yet functionally important modifications of cysteine.  

UK PubMed Central (United Kingdom)

The enhanced nucleophilicity and redox sensitivity of the thiol group renders cysteine residues susceptible to numerous electrophilic and oxidative posttranslational modifications to form disulfides, sulfenic acids, nitrosothiols and lipid-modified species. Outside of these well-characterized modifications of cysteine, there are reports of cysteine modification through phosphorylation, methylation and ubiquitination. Although these posttranslational modifications are highly abundant on other amino acids, they play a less pervasive role in cysteine biology. Despite the rarity of these modifications of cysteine, they have been shown to play critical roles in catalysis and regulation. Here we describe these rare posttranslational modifications of cysteine in detail, by describing their discovery and functional characterization on diverse proteins. Furthermore, we highlight potential proteomic tools that may aid in globally identifying these modifications to fully elucidate their abundance in biological systems.

Shannon DA; Weerapana E

2013-04-01

347

Pulse photolysis of NADH in the presence of cysteine  

International Nuclear Information System (INIS)

In the UV irradiation of NADH under anaerobic conditions, cysteine, which often acts as a radioprotective substance, has a sensitizing effect. With the aid of pulse photolysis, it was studied which reaction mechanisms in the presence or absence of cysteine are responsible for the damage to NADH in aqueous solution. In the absence of cysteine, the characteristic NADH absorption at 340 nm is reduced immediately after UV quanta have been absorbed by the adenine fraction of the molecules; in the presence of cysteine, a secondary reaction causes additional damage. The spectra of the intermediate products of NADH and cysteine have been recorded for different cysteine concentrations, and the reaction constants have been determined. These values suggest that the sensitizing effect is due to a reaction of NADH with radical anions produced by photolysis. (orig.)

1976-10-04

348

Modulation of ion transport across rat distal colon by cysteine.  

UK PubMed Central (United Kingdom)

The aim of this study was to identify the actions of stimulation of endogenous production of H(2)S by cysteine, the substrate for the two H(2)S-producing enzymes, cystathionine-?-synthase and cystathionine-?-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and ?-cyano-L-alanine, i.e., inhibitors of H(2)S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e., an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl(-) and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl(-) secretion, whereas Na cysteinate - after a transient inhibitory phase - activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K(+) channels. Indeed, after preinhibition of basolateral K(+) channels with tetrapentylammonium or Ba(2+), the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H(2)S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H(2)S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H(2)S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.

Pouokam E; Diener M

2012-01-01

349

Cysteine-containing peptides having antioxidant properties  

Energy Technology Data Exchange (ETDEWEB)

Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

Bielicki, John K. (Castro Valley, CA)

2009-10-13

350

Selective cleavage of glycyl bonds by papaya proteinase IV.  

UK PubMed Central (United Kingdom)

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO2 is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in beta-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.

Buttle DJ; Ritonja A; Pearl LH; Turk V; Barrett AJ

1990-01-01

351

Selective cleavage of glycyl bonds by papaya proteinase IV.  

Science.gov (United States)

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc-Ala-Ala-Gly-NHPhNO2 is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in beta-trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly-65 and Gly-23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV. PMID:2404797

Buttle, D J; Ritonja, A; Pearl, L H; Turk, V; Barrett, A J

1990-01-29

352

The precursor of secreted aspartic proteinase Sapp1p from Candida parapsilosis can be activated both autocatalytically and by a membrane-bound processing proteinase.  

Science.gov (United States)

Opportunistic pathogens of the genus Candida produce secreted aspartic proteinases (Saps) that play an important role in virulence. Saps are synthesized as zymogens, but cell-free culture supernatants of Candida spp. contain only mature Saps. To study the zymogen conversion, the gene encoding a precursor of C. parapsilosis proteinase Sapp1p was cloned, expressed in E. coli and the product was purified. When placed in acidic conditions, the precursor was autocatalytically processed, yielding an active proteinase. The self-activation proceeded through an intermediate product and the resulting enzyme was one amino acid shorter than the authentic enzyme. This truncation did not cause changes in proteinase activity or secondary structure compared to the authentic Sapp1p. Accurate cleavage of the pro-mature junction, however, required a processing proteinase. A crude membrane fraction prepared from C. parapsilosis cells contained an enzyme with Kex2-like activity, which processed the Sapp1p precursor at the expected site. The pro-segment appeared to be indispensable for Sapp1p to attain an appropriate structure. When expressed without the pro-segment, the Sapp1p mature domain was not active and had a lower content of alpha-helical conformation, as measured by circular dichroism. A similar effect was observed when a His(6)-tag was linked to the C-terminus of Sapp1p or its precursor. PMID:16201875

Dostál, Jirí; Dlouhá, Helena; Malon, Petr; Pichová, Iva; Hrusková-Heidingsfeldová, Olga

2005-08-01

353

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs).  

Science.gov (United States)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes. PMID:18627286

Ramsay, Andrew J; Reid, Janet C; Adams, Mark N; Samaratunga, Hemamali; Dong, Ying; Clements, Judith A; Hooper, John D

2008-06-01

354

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs).  

UK PubMed Central (United Kingdom)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.

Ramsay AJ; Reid JC; Adams MN; Samaratunga H; Dong Y; Clements JA; Hooper JD

2008-06-01

355

Ozone effects on inhibitors of human neutrophil proteinases  

Energy Technology Data Exchange (ETDEWEB)

The effects of ozone on human alpha 1-proteinase inhibitor (A-1-PI), alpha 1-antichymotrypsin (A-1-Achy), bronchial leukocyte proteinase inhibitor (BLPI), and Eglin C were studied using in vitro exposures in phosphate-buffered solutions. Following ozone exposure, inhibitory activities against human neutrophil elastase (HNE) and/or cathepsin G (Cat G) were measured. Exposure of A-1-PI to 50 mol O3/mol protein resulted in a complete loss of HNE inhibitory activity, whereas A-1-Achy lost only 50% of its Cat G inhibitory activity and remained half active even after exposure to 250 mol of O3. At 40 mol O3/mol protein, BLPI lost 79% of its activity against HNE and 87% of its Cat G inhibitory activity. Eglin C, a leech-derived inhibitor, lost 81% of its HNE inhibitory activity and 92% of its ability to inhibit Cat G when exposed to 40 mol O3/mol. Amino acid analyses of ozone-exposed inhibitors showed destruction of Trp, Met, Tyr, and His with as little as 10 mol O3/mol protein, and higher levels of O3 resulted in more extensive oxidation of susceptible residues. The variable ozone susceptibility of the different amino acid residues in the four proteins indicated that oxidation was a function of protein structure, as well as the inherent susceptibility of particular amino acids. Exposure of A-1-PI and BLPI in the presence of the antioxidants, Trolox C (water soluble vitamin E) and ascorbic acid (vitamin C), showed that antioxidant vitamins may protect proteins from oxidative inactivation by ozone. Methionine-specific modification of BLPI reduced its HNE and Cat G inhibitory activities. Two moles of N-chlorosuccinimide per mole of BLPI methionine caused an 80% reduction in activity against Cat G, but only a 40% reduction in HNE inhibitory activity.

Smith, C.E.; Stack, M.S.; Johnson, D.A.

1987-02-15

356

Novel inhibitors of procollagen C-proteinase. Part 2: glutamic acid hydroxamates.  

Science.gov (United States)

Glutamic acid derived hydroxamates were identified as potent and selective inhibitors of procollagen C-proteinase, an essential enzyme for the processing of procollagens to fibrillar collagens. Such compounds have potential therapeutic application in the treatment of fibrosis. PMID:12824039

Robinson, L A; Wilson, D M; Delaet, N G J; Bradley, E K; Dankwardt, S M; Campbell, J A; Martin, R L; Van Wart, H E; Walker, K A M; Sullivan, R W

2003-07-21

357

Purification and characterization of the heat-stable serine proteinase from Thermomonospora fusca YX.  

UK PubMed Central (United Kingdom)

The proteinase secreted from Thermomonospora fusca YX grown on cellulose was purified by (NH4)2SO4 fractionation and cation-exchange chromatography. The isolated proteinase readily hydrolysed several proteins and demonstrated activity towards casein from 35 to 95 degrees C (at pH 8.0) with maximum activity at 80 degrees C. It exhibited broad pH and ionic-strength optima centered at pH 9.0 and 0.2 M-NaCl respectively, and it retained high activity in the presence of 2% (w/v) SDS, 20 mM-dithiothreitol and 1.0 M-NaCl. The proteinase, which was fully inhibited by phenylmethanesulphonyl fluoride, had an Mr of 14,500 and an isoelectric point at 9.21. A measurement of proteinase thermal stability demonstrated a T50% (15 min) of 85 degrees C at pH 4.5.

Gusek TW; Kinsella JE

1987-09-01

358

Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin  

Energy Technology Data Exchange (ETDEWEB)

N-Acyl derivatives of 7-amino-4-trifluoromethylcoumarin (I) provide new and novel substrates for the assay of proteinases, allowing either direct fluorometric or photometric measurements. The yellow-green fluorescence of the free coumarin I makes it possible to visually observe hydrolysis of the arylamide bond, thus making it a very useful non-mutagenic fluorophore for the identification and quantification of proteinases whether in dynamic assay or in intact cells.

Smith, R.E.; Bissell, E.R.; Mitchell, A.R.; Pearson, K.W.

1980-01-01

359

[Properties of Bacillus pumilus subtilisin like proteinase secreted from recombinant strain on different growth stages].  

UK PubMed Central (United Kingdom)

Bacillus pumilus 3-19 glutamylendopeptidase has been isolated from culture liquid of Bacillus subtilis recombinant strain on different growth stages: growth retardation (early enzyme) and stationary phase (late enzyme). The effect of purified proteinase of different growth stages on insulin beta-chain, protein and oligopeptide substrates has been studied. Comparative study of physicochemical properties of early and late proteinases was carried out. Two protein fractions were different in catalytic characteristics and demonstrated different sensitivity to the presence of metal cations.

Balaban NP; Danilova IuV; Shamsutdinov TR; Mardanova AM; Cheremin AM; Rudenskaia GN; Sharipova MR

2013-01-01

360

Specific cleavage of hybrid proteins by proteinase encoded by the KEX2 gene.  

UK PubMed Central (United Kingdom)

A method for isolation of the KEX2-gene-encoded membrane-bound proteinase from alpha-cells of Saccharomyces cerevisiae yeast has been modified. The isolated enzyme hydrolyzes peptides and proteins with basic amino acid pairs which are cleaved at the C-ends of their peptide bonds. Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min. By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the membrane extract prepared according to the standard technique. This protocol decreases the number of steps required to isolate the enzyme. The effects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied. KEX2 proteinase can participate in peptide hormone processing because it cleaves human proinsulin at the peptide bond between Arg32 and Glu33. The KEX2 proteinase can specifically cleave large recombinant proteins, for example, a protein consisting of a gamma-interferon fragment linked to HIV1-proteinase via a Lys-Arg-containing peptide.

Bessmertnaya LYa; Loiko II; Goncharova TI; Ivanov NV; Rumsh LD; Antonov VK

1997-08-01

 
 
 
 
361

Purification and characterization of a proteinase from the probiotic Lactobacillus rhamnosus OXY.  

UK PubMed Central (United Kingdom)

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k(cat)/K(m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL(-1)) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+).

Wa?ko A; Kieliszek M; Targo?ski Z

2012-01-01

362

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme.  

UK PubMed Central (United Kingdom)

The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.

Schlick P; Kronovetr J; Hampoelz B; Skern T

2002-05-01

363

cDNA cloning and molecular modeling of procerain B, a novel cysteine endopeptidase isolated from Calotropis procera.  

UK PubMed Central (United Kingdom)

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed.

Singh AN; Yadav P; Dubey VK

2013-01-01

364

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits.  

Science.gov (United States)

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N-alpha-CBZ-amino acid-p-nitrophenyl esters, and the highest kcat/Km values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases. PMID:11594466

Obregón, W D; Arribére, M C; del Valle, S M; Liggieri, C; Caffini, N; Priolo, N

2001-05-01

365

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits.  

UK PubMed Central (United Kingdom)

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N-alpha-CBZ-amino acid-p-nitrophenyl esters, and the highest kcat/Km values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.

Obregón WD; Arribére MC; del Valle SM; Liggieri C; Caffini N; Priolo N

2001-05-01

366

Antibacterial Effect of Cysteine-Nitrosothiol and Possible Percursors Thereof  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The postulated intermediate of nitrite-myoglobin reaction, cysteine-nitrosothiol, was prepared and its antibacterial effect was tested on Salmonella strains, Streptococcus faecium, and spores and vegetative cells of Clostridium sporogenes. Cysteine-nitrosothiol showed a higher inhibitory effect than...

Incze, K.; Farkas, J.; Mihályi, Vilma; Zukál, E.

367

Rhodium-catalyzed cysteine modification with diazo reagents.  

UK PubMed Central (United Kingdom)

A simple rhodium(II) complex catalyzes cysteine modification with diazo reagents. The reaction is marked by clean cysteine selectivity and mild reaction conditions. The resulting linkage is significantly more stable in human plasma serum, when compared to common maleimide reagents.

Kundu R; Ball ZT

2013-05-01

368

CysView: protein classification based on cysteine pairing patterns  

Digital Repository Infrastructure Vision for European Research (DRIVER)

CysView is a web-based application tool that identifies and classifies proteins according to their disulfide connectivity patterns. It accepts a dataset of annotated protein sequences in various formats and returns a graphical representation of cysteine pairing patterns. CysView displays cysteine pa...

Lenffer, Johann; Lai, Paulo; El Mejaber, Wafaa; Khan, Asif M.; Koh, Judice L. Y.; Tan, Paul T. J.; Seah, Seng H.

369

Cysteine proteases of malaria parasites: targets for chemotherapy.  

UK PubMed Central (United Kingdom)

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.

Rosenthal PJ; Sijwali PS; Singh A; Shenai BR

2002-01-01

370

Centenary celebrations article: Cysteine proteases of human malaria parasites.  

UK PubMed Central (United Kingdom)

There is an urgent need for new drugs against malaria, which takes millions of lives annually. Cysteine proteases are potential new drug targets, especially when current drugs are showing resistance. Falcipains and vivapains are well characterized cysteine proteases of P. falciparum and P. vivax, respectively. Studies with cysteine protease inhibitors and manipulating cysteine proteases specific genes have suggested their roles in hemoglobin hydrolysis. In P. falciparum, falcipain-2 and falcipain-3 are major hemoglobinases that