RESUMO
The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K(i)=3.29 nM) and human cathepsin L (K(i)=3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.
Assuntos
Inibidores de Cisteína Proteinase/biossíntese , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Cistatinas Salivares/biossíntese , Triatoma/metabolismo , Triatoma/parasitologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Inibidores de Cisteína Proteinase/genética , Trato Gastrointestinal/metabolismo , Insetos Vetores/genética , Masculino , Dados de Sequência Molecular , Cistatinas Salivares/genética , Triatoma/genéticaRESUMO
Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight ß-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.
Assuntos
Inibidores de Cisteína Proteinase/genética , Entamoeba histolytica/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cristalografia , Cristalografia por Raios X/métodos , Cisteína Proteases/química , Cisteína Proteases/genética , Inibidores de Cisteína Proteinase/química , Entamoeba histolytica/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Peptídeo Hidrolases/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein.
Assuntos
Bauhinia/metabolismo , Besouros/crescimento & desenvolvimento , Inibidores Enzimáticos/metabolismo , Inseticidas/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Bauhinia/química , Bauhinia/genética , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Inibidores Enzimáticos/química , Genes de Plantas , Inseticidas/química , Calicreínas/antagonistas & inibidores , Larva/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Protozoários , Proteínas Recombinantes , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/química , Inibidores da Tripsina/genética , Inibidores da Tripsina/metabolismoRESUMO
It has been proposed that the natural cysteine peptidase inhibitor ICP of Leishmania mexicana protects the protozoan parasite from insect host proteolytic enzymes, thereby promoting survival. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. No significant differences were found between the wild-type parasites, two independently derived ICP-deficient mutants, or mutants overexpressing ICP; all lines developed similarly in the sand fly midgut and produced heavy late-stage infections. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of the midgut extracts in vitro. We conclude that ICP has no major role in promoting survival of L. mexicana in the vectorial part of its life cycle in L. longipalpis.
Assuntos
Inibidores de Cisteína Proteinase/fisiologia , Leishmania mexicana/patogenicidade , Proteínas de Protozoários/fisiologia , Psychodidae/parasitologia , Animais , Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/genética , Feminino , Interações Hospedeiro-Parasita , Proteínas de Insetos/fisiologia , Leishmania mexicana/genética , Proteínas de Protozoários/genética , Psychodidae/enzimologiaRESUMO
Leishmania mexicana cysteine peptidases (CPs) have been identified as important parasite virulence factors. More recently, a natural inhibitor of CPs (ICP) from L. mexicana has been characterized, and ICP mutants have been created. Infection of BALB/c mice with ICP null mutants or ICP reexpressing mutants resulted in nonhealing, progressively growing lesions albeit slightly attenuated compared with the growth of lesions produced by wild-type parasites. In contrast, BALB/c mice infected with mutants overexpressing ICP were able to significantly control lesion growth or heal. While BALB/c mice infected with wild-type parasites, ICP null mutants, or ICP reexpressing mutants produced significant antibody responses, including immunoglobulin E (IgE), no Th1 response, as indicated by antigen-induced splenocyte gamma interferon (IFN-gamma) production, could be demonstrated. In contrast, BALB/c mice infected with mutants overexpressing ICP produced significantly less antibody, particularly IgE, as well as significantly reduced splenocyte interleukin-4 and enhanced IFN-gamma production. BALB/c mice were able to resolve infection following infection with one ICP overexpressing clone, which was subsequently used for vaccination studies with BALB/c mice. However, no protection was afforded these mice when they were challenged with wild-type parasites. Nevertheless, two other mouse strains susceptible to L. mexicana, C3H and C57BL/6, vaccinated with overexpressing ICP mutants were able to control challenge infection associated with an enhanced Th1 response. This study confirms that L. mexicana CPs are virulence factors and that ICPs have therapeutic potential.
Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/biossíntese , Leishmania mexicana/imunologia , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/patologia , Células Th1/imunologia , Fatores de Virulência/antagonistas & inibidores , Animais , Anticorpos Antiprotozoários/sangue , Inibidores de Cisteína Proteinase/genética , Feminino , Deleção de Genes , Teste de Complementação Genética , Imunoglobulina E/sangue , Interferon gama/metabolismo , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Baço/imunologia , VirulênciaAssuntos
Inibidores de Cisteína Proteinase/genética , Evolução Molecular , Genoma de Planta , Populus/genética , Processamento Alternativo , Cistatinas/genética , Éxons , Etiquetas de Sequências Expressas , Duplicação Gênica , Genes de Plantas , Íntrons , Funções Verossimilhança , Filogenia , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Caspase 1 (CASP-1) inhibitors share sequence similarity to CASP-1 itself and are all mapped to chr11q22.3. Here we show that these inhibitors are all products of a series of gene duplications that occurred at this locus after the divergence between human and mouse. Surprisingly, stop codons originated independently in all duplicated copies to generate CARD-only proteins with inhibitory activity. We discuss this evolutionary model in the context of both neo- and subfunctionalization.
Assuntos
Proteínas de Transporte/genética , Inibidores de Cisteína Proteinase/genética , Evolução Molecular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Caspase 1/genética , Códon de Terminação , Éxons , Duplicação Gênica , Genes Duplicados , Humanos , Íntrons , Macaca mulatta , Dados de Sequência Molecular , Pan troglodytes/genéticaRESUMO
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.
Assuntos
Besouros/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Insetos/antagonistas & inibidores , Controle Biológico de Vetores , Proteínas de Protozoários/farmacologia , Animais , Besouros/efeitos dos fármacos , Besouros/fisiologia , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Trato Gastrointestinal/enzimologia , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
ICP is a chagasin-family natural tight binding inhibitor of Clan CA, family C1 cysteine peptidases (CPs). We investigated the role of ICP in Trypanosoma brucei by generating bloodstream form ICP-deficient mutants (Deltaicp). A threefold increase in CP activity was detected in lysates of Deltaicp, which was restored to the levels in wild type parasites by re-expression of the gene in the null mutant. Deltaicp displayed slower growth in culture and increased resistance to a trypanocidal synthetic CP inhibitor. More efficient exchange of the variant surface glycoprotein (VSG) to procyclin during differentiation from bloodstream to procyclic form was observed in Deltaicp, a phenotype that was reversed in the presence of synthetic CP inhibitors. Furthermore, we showed that degradation of anti-VSG IgG is abolished when parasites are pretreated with synthetic CP inhibitors, and that parasites lacking ICP degrade IgG more efficiently than wild type. In addition, Deltaicp reached higher parasitemia than wild type parasites in infected mice, suggesting that ICP modulates parasite infectivity. Taken together, these data suggest that CPs of T. brucei bloodstream form play a role in surface coat exchange during differentiation, in the degradation of internalized IgG and in parasite infectivity, and that their function is regulated by ICP.
Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Regulação da Expressão Gênica , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/patogenicidade , Animais , Inibidores de Cisteína Proteinase/genética , Deleção de Genes , Camundongos , Camundongos Endogâmicos BALB C , Parasitemia/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Tripanossomíase Africana/parasitologia , VirulênciaRESUMO
A cDNA, encoding a cysteine protease inhibitor (AhCPI), was isolated from an immature seed cDNA library of grain amaranth (Amaranthus hypochondriacus L.) and characterized. It encoded a polypeptide of 247 amino acids (aa), including a putative N-terminal signal peptide. Other relevant regions found in its sequence included the G and PW conserved aa motifs, the consensus LARFAV sequence for phytocystatins and the reactive site QVVAG. The predicted aa sequence for AhCPI showed a significant homology to other plant cystatins. Gene expression analyses indicated that AhCPI was constitutively expressed in mature seeds, and gradually decreased during germination. In vegetative tissues, AhCPI was expressed in the radicle and hypocotyls of seedlings and in the stems and roots of young plantlets. Its expression in roots and stems increased substantially in response to water deficit, salinity-, cold- and heat-stress, whereas heat-stress induced a rapid and transient accumulation of AhCPI transcripts in leaves. The results obtained were suggestive of multiple roles for AhCPI in grain amaranth, acting as a regulator of seed germination and as a protective agent against diverse types of abiotic stress, which induced this gene in a tissue- and stress-specific manner. The work herewith described reports a novel, and apparently, single cystatin protein in which, in agreement with other plant model systems, could have a regulatory role in germination, and further expands previous findings linking the accumulation of protease inhibitors, mostly of the serine proteinase type, with protection against (a)biotic stress in A. hypochondriacus.
Assuntos
Amaranthus/genética , Cistatinas/genética , DNA Complementar/genética , Germinação/genética , Proteínas de Plantas/genética , Amaranthus/efeitos dos fármacos , Amaranthus/crescimento & desenvolvimento , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , Inibidores de Cisteína Proteinase/genética , DNA Complementar/química , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Caules de Planta/efeitos dos fármacos , Caules de Planta/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.
Assuntos
Besouros/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , DNA Complementar/genética , Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxinas/metabolismoRESUMO
Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.
Assuntos
Cistatinas/química , Fabaceae/química , Modelos Moleculares , Proteínas de Plantas/química , Gorgulhos , Agricultura/métodos , Sequência de Aminoácidos , Animais , Cistatinas/genética , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Fabaceae/genética , Fabaceae/metabolismo , Dados de Sequência Molecular , Controle Biológico de Vetores/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Engenharia de Proteínas/métodos , Estrutura Secundária de ProteínaRESUMO
Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
Assuntos
Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Evolução Molecular , Leishmania mexicana/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Inibidores de Cisteína Proteinase/classificação , Inibidores de Cisteína Proteinase/genética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Papaína/química , Papaína/metabolismo , Dobramento de Proteína , Alinhamento de SequênciaRESUMO
Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.
Assuntos
Inibidores de Cisteína Proteinase/genética , Proteínas de Plantas/genética , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Sequência de Bases , Bauhinia , Sítios de Ligação , Catepsina L , Catepsinas/antagonistas & inibidores , Dicroísmo Circular , Clonagem Molecular , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , DNA Complementar/isolamento & purificação , DNA de Plantas/análise , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas de Protozoários , RNA de Plantas/análise , Proteínas Recombinantes/farmacologia , Sementes/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Inibidores de Serina Proteinase/farmacologiaRESUMO
T-kininogen (T-KG) is a precursor of T-kinin, the most abundant kinin in rat serum, and also acts as a strong and specific cysteine proteinase inhibitor. Its expression is strongly induced during aging in rats, and expression of T-KG in Balb/c 3T3 fibroblasts results in inhibition of cell proliferation. However, T-KG is a serum protein produced primarily in the liver, and thus, most cells are only exposed to the protein from the outside. To test the effect of T-KG on fibroblasts exposed to exogenous T-KG, we purified the protein from the serum of K-kininogen-deficient Katholiek rats. In contrast to the results obtained by transfection, exposure of Balb/c 3T3 fibroblasts to exogenously added T-KG leads to a dose-dependent increase in [3H]-thymidine incorporation. This response does not require kinin receptors, but it is clearly mediated by activation of the ERK pathway. As a control, we repeated the transfection experiments, using a different promoter. The results are consistent with our published data showing that, under these circumstances, T-KG inhibits cell proliferation. We conclude that T-KG exerts opposite effects on fibroblast proliferation, depending exclusively on the way that it is administered to the cells (transfection versus exogenous addition).
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Cininogênios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Células 3T3 BALB , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cininogênios/genética , Cininogênios/metabolismo , Camundongos , Ratos , TransfecçãoRESUMO
Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.
Assuntos
Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase/genética , Peptídeos/genética , Doenças das Plantas/parasitologia , Tylenchoidea/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , DNA Complementar/genética , DNA de Helmintos/genética , Feminino , Dados de Sequência Molecular , Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Tylenchoidea/genéticaRESUMO
The complete sequence of SmCys, a cystatin expressed by Schistosoma mansoni, was obtained. Constructs of SmCys consisting of deletions of 10 and 20 amino acid residues from the N-terminal of the full length recombinant protein, were cloned in the pQE-30 vector, expressed in Escherichia coli and assayed for inhibitory activity against papain. Kinetic analysis showed that SmCys -10 and SmCys -20 had K(i) values of 0.7391 and 4.9154, respectively, as compared to 0.0647, displayed by the full length recombinant. Protease inhibition by SmCys was also observed in vivo. When the recombinant products were incubated during 7 days with live schistosomula in the presence of red blood cells, only the full length product could completely inhibit the formation of haemozoin, a dark pigment formed as a by-product of haemoglobin digestion. The sequence data of the recombinant SmCys proteins were used for the construction of molecular models, which were then subjected to molecular dynamics for 2ns. In comparison to the full length, the models corresponding to the truncated constructs, showed a distinctive change on the surface charge distribution. This parameter was more pronounced in SmCys -20, which also displayed a significant displacement of the inhibitory domain, a result which could explain the kinetic data in terms of the loss of attachment sites. These changes correlated well with the progressive lack of inhibition observed for the recombinant deletion constructs, in vitro and in vivo.
Assuntos
Cistatinas/química , Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/química , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Simulação por Computador , Cistatinas/genética , Cistatinas/isolamento & purificação , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/isolamento & purificação , Inibidores de Cisteína Proteinase/farmacologia , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/fisiologia , Hemeproteínas/metabolismo , Hemoglobinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Papaína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Schistosoma mansoni/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.
Assuntos
Feminino , Animais , Cisteína Endopeptidases/genética , Doenças das Plantas/parasitologia , Inibidores de Cisteína Proteinase/genética , Peptídeos/genética , Tylenchoidea/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cisteína Endopeptidases/metabolismo , DNA Complementar/genética , DNA de Helmintos/genética , Inibidores de Cisteína Proteinase/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Peptídeos/metabolismo , Tylenchoidea/genéticaRESUMO
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.
Assuntos
Besouros/enzimologia , Cistatinas/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Animais , Besouros/embriologia , Cistatinas/genética , Cistatinas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Estrutura Terciária de ProteínaRESUMO
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.