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1.
J Biol Chem ; 299(6): 104801, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37164157

RESUMO

Papain-like cysteine peptidases form a big and highly diverse superfamily of proteins involved in many important biological functions, such as protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling. High sequence and structure diversity observed within these proteins hinders their comprehensive classification as well as the identification of new representatives. Moreover, in general protein databases, many families already classified as papain like lack details regarding their mechanism of action or biological function. Here, we use transitive remote homology searches and 3D modeling to newly classify 21 families to the papain-like cysteine peptidase superfamily. We attempt to predict their biological function and provide structural characterization of 89 protein clusters defined based on sequence similarity altogether spanning 106 papain-like families. Moreover, we systematically discuss observed diversity in sequences, structures, and catalytic sites. Eventually, we expand the list of human papain-related proteins by seven representatives, including dopamine receptor-interacting protein 1 as potential deubiquitinase, and centriole duplication regulating CEP76 as retaining catalytically active peptidase-like domain. The presented results not only provide structure-based rationales to already existing peptidase databases but also may inspire further experimental research focused on peptidase-related biological processes.


Assuntos
Cisteína Proteases , Papaína , Humanos , Domínio Catalítico , Centríolos/metabolismo , Cisteína Proteases/química , Cisteína Proteases/classificação , Cisteína Proteases/metabolismo , Enzimas Desubiquitinantes/metabolismo , Modelos Moleculares , Papaína/química , Papaína/classificação , Bases de Dados de Proteínas
2.
Curr Genet ; 65(1): 293-300, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30167777

RESUMO

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.


Assuntos
Botrytis/genética , Cisteína Proteases/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Sequência de Aminoácidos , Autofagia/genética , Botrytis/metabolismo , Botrytis/patogenicidade , Cucumis sativus/microbiologia , Cisteína Proteases/classificação , Cisteína Proteases/metabolismo , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Mutação , Filogenia , Folhas de Planta/microbiologia , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Virulência/genética
4.
Infect Genet Evol ; 51: 33-40, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28286139

RESUMO

Fasciola gigantica is regarded as the major liver fluke causing fasciolosis in livestock in tropical countries. Despite the significant economic and public health impacts of F. gigantica there are few studies on the pathogenesis of this parasite and our understanding is further limited by the lack of genome and transcriptome information. In this study, de novo Illumina RNA sequencing (RNA-seq) was performed to obtain a comprehensive transcriptome profile of the juvenile (42days post infection) and adult stages of F. gigantica. A total of 49,720 unigenes were produced from juvenile and adult stages of F. gigantica, with an average length of 1286 nucleotides (nt) and N50 of 2076nt. A total of 27,862 (56.03%) unigenes were annotated by BLAST similarity searches against the NCBI non-redundant protein database. Because F. gigantica needs to feed and/or digest host tissues, some proteases (including cysteine proteases and aspartic proteases), which play a role in the degradation of host tissues (protein), have been paid more attention in the present study. A total of 6511 distinct genes were found differentially expressed between juveniles and adults, of which 3993 genes were up-regulated and 2518 genes were down-regulated in adults versus juveniles, respectively. Moreover, stage-specific differentially expressed genes were identified in juvenile (17,009) and adult (6517) F. gigantica. The significantly divergent pathways of differentially expressed genes included cAMP signaling pathway (226; 4.12%), proteoglycans in cancer (256; 4.67%) and focal adhesion (199; 3.63%). The transcription pattern also revealed two egg-laying-associated pathways: cGMP-PKG signaling pathway and TGF-ß signaling pathway. This study provides the first comparative transcriptomic data concerning juvenile and adult stages of F. gigantica that will be of great value for future research efforts into understanding parasite pathogenesis and developing vaccines against this important parasite.


Assuntos
Fasciola/genética , Fasciolíase/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Proteínas de Helminto/genética , Redes e Vias Metabólicas/genética , Transcriptoma , Animais , Ácido Aspártico Proteases/classificação , Ácido Aspártico Proteases/genética , Búfalos , Cisteína Proteases/classificação , Cisteína Proteases/genética , Bases de Dados Genéticas , Fasciola/isolamento & purificação , Fasciola/metabolismo , Fasciolíase/parasitologia , Perfilação da Expressão Gênica , Ontologia Genética , Proteínas de Helminto/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Transdução de Sinais
5.
Plant Physiol ; 168(4): 1462-75, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26048883

RESUMO

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.


Assuntos
Cisteína Proteases/metabolismo , Corantes Fluorescentes/química , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Cisteína Endopeptidases/classificação , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cisteína Proteases/classificação , Cisteína Proteases/genética , Corantes Fluorescentes/síntese química , Germinação/genética , Modelos Químicos , Estrutura Molecular , Mutação , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/crescimento & desenvolvimento , Nicotiana/genética , Nicotiana/metabolismo
6.
J Egypt Soc Parasitol ; 43(3): 787-96, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24640878

RESUMO

Fasciolosis caused by Fasciola gigantica is one of the major public health problems in the world including Egypt. Immunodiagnostic methods are more applicable for their better sensitivity and specificity than other methods. The present study was conducted to cysteine proteinase (CP) antigens of F. gigantica in IgG-ELISA to diagnose human fasciolosis. IgG-ELISA with 2 cysteine proteinases of 27 kDa (Fas1) and 29 kDa (Fas2), obtained from the regurgitated materials of adult worms, were evaluated using serum samples from 90 Egyptian patients infected with F. gigantica, 55 patients with other parasitic infections and 50 healthy volunteers. The diagnostic sensitivity and specificity of Fas1 for detection of F. gigantica infection were 91.1% and 89.1%, respectively. The positivity of the assay was 95%. The positive and negative predicted values were 91% and 86%, respectively. These data suggest that IgG-ELISA with Fas1 is highly sensitive and specific assay and could be used for the immunodiagnosis of human fasciolosis.


Assuntos
Antígenos de Helmintos/imunologia , Cisteína Proteases/classificação , Fasciola/classificação , Fasciolíase/parasitologia , Animais , Cisteína Proteases/genética , Cisteína Proteases/metabolismo , Egito/epidemiologia , Fasciolíase/epidemiologia , Fezes/parasitologia , Regulação da Expressão Gênica , Humanos , Contagem de Ovos de Parasitas
7.
J Proteome Res ; 11(12): 5983-93, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23082957

RESUMO

Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.


Assuntos
Besouros/enzimologia , Cistatinas/metabolismo , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Animais , Cisteína Proteases/classificação , Ensaios Enzimáticos , Proteínas de Insetos/metabolismo , Larva/enzimologia , Modelos Biológicos , Simulação de Acoplamento Molecular , Complexos Multiproteicos/metabolismo , Mutação , Filogenia , Proteínas de Plantas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Proteômica/métodos , Relação Estrutura-Atividade , Especificidade por Substrato
8.
Integr Biol (Camb) ; 4(11): 1351-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23001143

RESUMO

UNLABELLED: There has been a revival of interest in Cysteine protease for Visceral Leishmaniasis (VL) attributed to massive outbreaks of leishmaniasis in the tropical region. The cysteine protease database (CPDB) was designed to find data related to cysteine protease (CP) of different species of Leishmania and Trypanosoma brucei in a single platform. This has reflected in substantial increase in the submission of Leishmania genome sequences to NCBI (National Center for Biotechnology Information) database. The CPDB database aims to provide a summary of data analysis, such as physiochemical and molecular properties, proteolytic cleavage sites, classification into functional families using SVMProt and other ExPASy tools. The main aim of this database is to provide different protein inhibitors of cysteine protease groups that were collected from literature and make available their 3-D structures through JMol with JAVA platform. These CP inhibitors are freely downloadable and also have added links for functional analyses of other proteins, which is helpful for users. All this information in CPDB, a single platform, will prove to be of great help for researchers who are involved in drug discovery and analysis of other physiochemical and molecular properties of the protein. AVAILABILITY: the database is available for free at.


Assuntos
Cisteína Proteases/genética , Bases de Dados de Compostos Químicos , Leishmania/enzimologia , Leishmania/genética , Animais , Cisteína Proteases/química , Cisteína Proteases/classificação , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Descoberta de Drogas , Genes de Protozoários , Humanos , Leishmania/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Especificidade da Espécie , Biologia de Sistemas
9.
Exp Parasitol ; 127(1): 184-94, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20655912

RESUMO

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.


Assuntos
Babesia/enzimologia , Babesiose/veterinária , Doenças dos Bovinos/parasitologia , Cisteína Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Babesia/classificação , Babesia/genética , Babesiose/parasitologia , Sequência de Bases , Bovinos , Clonagem Molecular , Cisteína Proteases/química , Cisteína Proteases/classificação , Cisteína Proteases/genética , Evolução Molecular , Regulação da Expressão Gênica/genética , Genoma de Protozoário , Filogenia , Alinhamento de Sequência/veterinária , Theileria/classificação , Theileria/enzimologia , Theileria/genética
10.
Transbound Emerg Dis ; 57(1-2): 68-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20537109

RESUMO

Papain-like cysteine proteases (CP) have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. One gene was identified by sequence similarity search to be homologous to the CP family in the ongoing Babesia bigemina genome sequencing project database. The newly identified CP gene, called babesipain-1, was cloned and expressed as a fusion protein, and the effect of different inhibitors on proteolytic activity was tested. A series of new artemisinin-vinyl sulfone hybrid molecules were tested as inhibitors being effective on the range of 0.3-30 microm, depending on the core-containing molecule.


Assuntos
Antiprotozoários/farmacologia , Babesia/metabolismo , Cisteína Proteases/classificação , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
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