RESUMO
The regulation of protease activity is a critical factor for the physiological balance during plant growth and development. Among the proteins involved in controlling protease activity are the cystatins, well-described inhibitors of cysteine proteases present in viruses, bacteria and most Eukaryotes. Plant cystatins, commonly called phytocystatins, display unique structural and functional diversity and are classified according to their molecular weight as type-I, -II, and -III. Their gene structure is highly conserved across Viridiplantae and provides insights into their evolutionary relationships. Many type-I phytocystatins with introns share sequence similarities with type-II phytocystatins. New data shows that they could have originated from recent losses of the carboxy-terminal extension present in type-II phytocystatins. Intronless type-I phytocystatins originated from a single event shared by flowering plants. Pieces of evidence show multiple events of gene duplications, intron losses, and gains throughout the expansion and diversity of the phytocystatin family. Gene duplication events in Gymnosperms and Eudicots resulted in inhibitors with amino acid substitutions that may modify their interaction with target proteases and other proteins. This review brings a phylogenomic analysis of plant cystatin evolution and contributes to a broader understanding of their origins. A complete functional genomic analysis among phytocystatins and their roles in plant development and responses to abiotic and biotic stresses remains a question to be fully solved.
Assuntos
Cistatinas , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/química , Duplicação Gênica , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Estresse FisiológicoRESUMO
Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.
Assuntos
Inibidores de Cisteína Proteinase/química , Sanguessugas/química , Saccharomycetales/metabolismo , Animais , Catepsina L , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , DNA Complementar , Humanos , Sanguessugas/genética , Proteínas RecombinantesRESUMO
Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.
Assuntos
Cistatinas/química , Osteoblastos/citologia , beta Catenina/metabolismo , Células 3T3 , Animais , Anti-Inflamatórios/química , Adesão Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Citrus sinensis , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Citoesqueleto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteogênese , Compostos Fitoquímicos , CicatrizaçãoRESUMO
Phytocystatins are a family of plant cysteine-protease inhibitors of great interest due to their biotechnological application in culture improvement. It was shown that their expression in plants increases resistance to herbivory by insects and improves tolerance to both biotic and abiotic stress factors. In this work, owing to the economical relevance of the source organism, a phytocystatin from hop (Humulus lupulus), Hop1, was produced by heterologous expression in E. coli Lemo21 (DE3) cultivated in auto-inducing ZYM-5052 medium and purified by immobilized metal ion affinity and size exclusion chromatography. Thermal denaturation assays by circular dichroism showed that Hop1 exhibited high melting temperatures ranging from 82 °C to 85 °C and high thermal stability at a wide pH range, with ΔG25's higher than 12 kcal/mol. At 20 °C and pH 7.6, the dimeric conformation of the protein is favored according to size exclusion chromatography and analytical ultracentrifugation data, although monomers and higher order oligomers could still be detected in a lesser extent. The crystal structure of Hop1 was solved in the space groups P 2 21 21 and C 2 2 21 at resolutions of 1.80 Å and 1.68 Å, respectively. In both models, Hop1 is folded as a domain-swapped dimer where the first inhibitory loop undergoes a significant structural change and interacts with their equivalent from the other monomer forming a long antiparallel beta strand, leading to loss of inhibitory activity.
Assuntos
Cistatinas/química , Inibidores de Cisteína Proteinase/química , Humulus/química , Proteínas de Plantas/química , Clonagem Molecular , Cristalografia por Raios X , Cistatinas/genética , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.
Assuntos
Aedes , Catepsina L , Cistatinas , Vírus da Dengue/metabolismo , Proteínas de Insetos , Aedes/enzimologia , Aedes/genética , Aedes/virologia , Animais , Catepsina L/química , Catepsina L/genética , Catepsina L/metabolismo , Linhagem Celular , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismoRESUMO
Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (pâ¯<â¯0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (pâ¯<â¯0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
Assuntos
Antígenos de Diferenciação/biossíntese , Citrus/química , Cistatinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Animais , Cistatinas/química , Humanos , Macrófagos/patologia , Masculino , Camundongos , Proteínas de Plantas/química , Células RAW 264.7 , Ratos , Ratos WistarRESUMO
Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.
Assuntos
Antimaláricos/farmacologia , Cistatinas/farmacologia , Cisteína Proteases/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Proteínas de Plantas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Cistatinas/química , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/genética , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/isolamento & purificação , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Eritrócitos/fisiologia , Hemeproteínas/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas de Plantas/química , Plasmodium falciparum/enzimologiaRESUMO
Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.
Assuntos
Cistatinas/genética , Inibidores de Cisteína Proteinase/farmacologia , Fasciola hepatica/genética , Proteínas de Helminto/genética , Sequência de Aminoácidos , Animais , Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Bovinos , Cistatinas/química , Cistatinas/metabolismo , Escherichia coli/genética , Fasciola hepatica/enzimologia , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Humanos , Organismos Geneticamente Modificados , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.
Assuntos
Cistatinas/química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Escherichia coli/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Temperatura , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/isolamento & purificação , Estabilidade Enzimática , Expressão Gênica , Modelos Moleculares , Proteínas de Plantas/isolamento & purificação , Domínios Proteicos , Análise de Sequência , Água/químicaRESUMO
Cystatins are cysteine peptidase inhibitors that in ticks mediate processes such as blood feeding and digestion. The ixodid tick Ixodes persulcatus is endemic to the Eurasia, where it is the principal vector of Lyme borreliosis. To date, no I. persulcatus cystatin has been characterized. In the present work, we describe three novel cystatins from I. persulcatus, named JpIpcys2a, JpIpcys2b and JpIpcys2c. In addition, the potential of tick cystatins as cross-protective antigens was evaluated by vaccination of hamsters using BrBmcys2c, a cystatin from Rhipicephalus microplus, against I. persulcatus infestation. Sequence analysis showed that motifs that are characteristic of cystatins type 2 are fully conserved in JpIpcys2b, while mutations are present in both JpIpcys2a and JpIpcys2c. Protein-protein docking simulations further revealed that JpIpcys2a, JpIpcys2b and JpIpcys2c showed conserved binding sites to human cathepsins L, all of them covering the active site cleft. Cystatin transcripts were detected in different I. persulcatus tissues and instars, showing their ubiquitous expression during I. persulcatus development. Serological analysis showed that although hamsters immunized with BrBmcys2c developed a humoral immune response, this response was not adequate to protect against a heterologous challenge with I. persulcatus adult ticks. The lack of cross-protection provided by BrBmcys2c immunization is perhaps linked to the fact that cystatins cluster into multigene protein families that are expressed differentially and exhibit functional redundancy. How to target such small proteins that are secreted in low quantities remains a challenge in the development of suitable anti-tick vaccine antigens.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cistatinas/química , Cistatinas/genética , Ixodes/metabolismo , Infestações por Carrapato/prevenção & controle , Animais , Anticorpos/sangue , Anticorpos/imunologia , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/isolamento & purificação , Sítios de Ligação , Catepsina L/química , Cricetinae , Humanos , Imunidade Humoral , Ixodes/imunologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Família Multigênica , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Rhipicephalus/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Phytocystatins encompass a family of plant competitive cysteine proteinase inhibitors. They are encoded by part of a conserved monophyletic group of genes that are found in all eukaryotes. The primary targets of phytocystatins are papain-like cysteine proteinases. However, a group of larger phytocystatins is also able to inhibit proteinases such as legumains. Phytocystatins have been implicated in several physiological processes and act within an intricate proteolytic regulatory network. The present work characterizes the gene family of rice phytocystatins, which contains twelve genes with different features. Phylogenetic analyses cluster rice phytocystatins into three main groups. Group 1 is composed of OcI, OcIII and OcXII and is nearly ubiquitous and highly expressed in plants under normal and stressed conditions including salt, wounding, ABA or a fungal elicitor such as chitosan. Rice phytocystatins can contribute to plant senescence and may exhibit an inverse correlation between their gene expression and the activities of their target proteinases. This work contributes to clarifying the roles of individual phytocystatin genes in plant processes such as germination and response to environmental stresses.
Assuntos
Cistatinas/metabolismo , Família Multigênica , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Cistatinas/química , Cistatinas/genética , Expressão Gênica , Oryza/genética , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMO
Various classes of endopeptidases and their inhibitors facilitate blood feeding and digestion in ticks. Cystatins, a family of tight-binding and reversible inhibitors of cysteine endopeptidases, have recently been found in several tick tissues. Moreover, vaccine trials using tick cystatins have been found to induce protective immune responses against tick infestation. However, the mode of action of tick cystatins is still poorly understood, limiting the elucidation of their physiological role. Against this background, we have investigated sequence characteristics and immunogenic properties of 5 putative cystatins from Rhipicephalus (Boophilus) microplus from Brazil and Uruguay. The similarity of the deduced amino acid sequences among cystatins from the Brazilian tick strain was 27-42%, all of which had a secretory signal peptide. The cystatin motif (QxVxG), a glycine in the N-terminal region, and the PW motif in the second hairpin loop in the C-terminal region are highly conserved in all 5 cystatins identified in this study. Four cysteine residues in the C terminus characteristic of type 2 cystatins are also present. qRT-PCR revealed differential expression patterns among the 5 cystatins identified, as well as variation in mRNA transcripts present in egg, larva, gut, salivary glands, ovary, and fat body tissues. One R. microplus cystatin showed 97-100% amino acid similarity between Brazilian and Uruguayan isolates. Furthermore, by in silico analysis, antigenic amino acid regions from R. microplus cystatins showed high degrees of homology (54-92%) among Rhipicephalus spp. cystatins. Three Brazilian R. microplus cystatins were expressed in Escherichia coli, and immunogenicity of the recombinant proteins were determined by vaccinating mice. Western blotting using mice sera indicated cross-reactivity between the cystatins, suggesting shared epitopes. The present characterization of Rhipicephalus spp. cystatins represents an empirical approach in an effort to evaluate the physiological role of cystatins in a larger context of targeting them for use in future tick control strategies.
Assuntos
Doenças dos Bovinos/parasitologia , Cistatinas/química , Cistatinas/imunologia , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Sequência de Aminoácidos , Animais , Brasil , Bovinos , Doenças dos Bovinos/imunologia , Biologia Computacional , Cistatinas/genética , DNA Complementar/química , DNA Complementar/genética , Feminino , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhipicephalus/química , Rhipicephalus/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologiaRESUMO
As Aedes aegypti transmits the etiologic agents of both yellow and dengue fever; vector control is considered essential to minimise their incidence. The aim of this work was to identify the component of Carica papaya seed toxic to A. aegypti, and the identification of tegupain, the enzyme that generates it. Aqueous extracts (1%, w/v) of the seed tegument and cotyledon of C. papaya are not larvicidal isolately. However, a mixture of 17µgmL(-1) tegument extract and 27µgmL(-1) cotyledon extract caused 100% larval mortality in a bioassay. The mixture was no longer larvicidal after the tegument extract was pre-treated at 100°C for 10min. The enzyme tegupain efficiently hydrolysed the substrate Z-Phe-Arg-pNan (Km 58.8µM, Kcat 28020s(-1), Kcat/Km 5×10(8)M(-1) s(-1)), and its activity increased with 2mM dithiothreitol (DTT), at 37°C, pH 5.0. The chelating agent EDTA did not modify the enzyme activity. Inhibition of tegupain by cystatin (Kiapp 2.43nM), E64 (3.64nM, 83% inhibition), and the propeptide N-terminal sequence indicate that the toxic activity is due to a novel cysteine proteinase-like enzyme, rendered active upon the hydrolysis of a cotyledon component of C. papaya seeds.
Assuntos
Aedes/efeitos dos fármacos , Carica/química , Extratos Vegetais/toxicidade , Proteínas de Plantas/toxicidade , Aedes/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Cotilédone/química , Cistatinas/química , Cistatinas/metabolismo , Concentração de Íons de Hidrogênio , Larva/efeitos dos fármacos , Dados de Sequência Molecular , Extratos Vegetais/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Ligação Proteica , Sementes/química , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The three-dimensional structure of canecystatin-1, a potent inhibitor of cysteine proteases from sugarcane (Saccharum officinarum), has been solved in two different crystal forms. In both cases, it is seen to exist as a domain-swapped dimer, the first such observation for a cystatin of plant origin. Size exclusion chromatography and multidimensional NMR spectroscopy show the dimer to be the dominant species in solution, despite the presence of a measurable quantity of monomer undergoing slow exchange. The latter is believed to be the active species, whereas the domain-swapped dimer is presumably inactive, as its first inhibitory loop has been extended to form part of a long ß-strand that forms a double-helical coiled coil with its partner from the other monomer. A similar structure is observed in human cystatin C, but the spatial disposition of the two lobes of the dimer is rather different. Dimerization is presumably a mechanism by which canecystatin-1 can be kept inactive within the plant, avoiding the inhibition of endogenous proteases. The structure described here provides a platform for the rational design of specific cysteine protease inhibitors for biotechnological applications.
Assuntos
Cistatinas/química , Proteínas de Plantas/química , Saccharum , Cristalografia por Raios X , Cistatinas/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
Cystatins are cysteine proteases inhibitors that are widely distributed among insects, mammalians and plants. Here we report the complete resonance assignment of canecystatin-1 from Saccharum officinarum obtained by heteronuclear multidimensional high-resolution nuclear magnetic resonance spectroscopy. The consensus chemical shift index was calculated and showed the presence of one α-helix (residues 27-43) and three ß-strands (residues 48-74, 78-89 and 94-104), a secondary structure pattern that suggests a domain-swapped structure as presented by stefin B and human cystatin C, opposed to the monomeric structure yet found in other phytocystatins like oryza and pineapple cystatin.
Assuntos
Cistatinas/química , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Saccharum/metabolismo , Humanos , Estrutura Secundária de ProteínaRESUMO
The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.
Assuntos
Cistatinas/análise , Cistatinas/química , Carrapatos/metabolismo , Sequência de Aminoácidos , Animais , Cistatinas/genética , Cistatinas/metabolismo , Dados de Sequência Molecular , Peso MolecularRESUMO
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
A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.
Assuntos
Cistatinas/química , Inibidores de Cisteína Proteinase/química , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Cistatinas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/química , Trypanosoma cruzi/metabolismoRESUMO
Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.
Assuntos
Cistatinas/biossíntese , Cistatinas/isolamento & purificação , Escherichia coli , Proteínas de Plantas/biossíntese , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Cromatografia de Afinidade , Cistatinas/química , Expressão Gênica , Papaína/antagonistas & inibidores , Papaína/química , Proteínas de Plantas/química , Proteínas Recombinantes/químicaRESUMO
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 =3.3nM) 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=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), 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.