RESUMO
Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.
Assuntos
Canavalia/enzimologia , Inseticidas/isolamento & purificação , Urease/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Inseticidas/química , Inseticidas/toxicidade , Dados de Sequência Molecular , Proteínas de Plantas , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Urease/genética , Urease/isolamento & purificação , Urease/toxicidadeRESUMO
Chagasin, a protein from Trypanosoma cruzi, is the first member of a new family of tight binding cysteine protease inhibitors [Monteiro, A.C.S., Abrahamson, M., Lima, A.P.C., Vannier-Santos, M.A. and Scharfstein, J. (2001) J. Cell Sci., in press] [corrected]. Despite its lack of significant sequence identity with known proteins, convincing structural models, using variable light chain templates, could be constructed on the basis of threading results. Experimental support for the final structure came from inhibition data for overlapping oligopeptides spanning the chagasin sequence. Chagasin therefore exemplifies a new protease inhibitor structural class and a new natural use for an immunoglobulin-like domain. Limited sequence resemblance suggests that chagasin may represent the result of a rare horizontal gene transfer from host to parasite.
Assuntos
Inibidores de Cisteína Proteinase/química , Transferência Genética Horizontal , Imunoglobulinas/química , Dobramento de Proteína , Proteínas de Protozoários/química , Trypanosoma cruzi/química , Sequência de Aminoácidos , Animais , Inibidores de Cisteína Proteinase/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Homologia de Sequência de AminoácidosRESUMO
The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.
Assuntos
Coenzimas/metabolismo , Geobacillus stearothermophilus/enzimologia , Complexos Multienzimáticos/metabolismo , Fosfoglicerato Mutase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência Consenso , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Fosfoglicerato Mutase/química , Fosfoproteínas Fosfatases/química , Filogenia , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree-frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier-transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti-parallel beta-sheet. A conformational variation was observed under experimental conditions mimicking a membrane-like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.
Assuntos
Antibacterianos/farmacologia , Anuros/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Membrana Celular , Dicroísmo Circular , Dimerização , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Dominant plant resistance genes are involved in the protection of plants against a wide variety of pathogens. Sequence analysis has revealed a variety of classes, often having domains in common. One commonly found region has come to be known as a putative nucleotide-binding site (NBS) due to the simple presence of sequence motifs. Until now, no experimental evidence has supported this idea. Here we suggest, as an alternative hypothesis, that part of this region is structurally homologous to the receiver domain common to many proteins of His-Asp phosphotransfer pathways. This conclusion is based on sequence analysis, threading experiments, and the construction of a molecular model of one domain that performs well against structure validation tools. The new hypothesis, in contrast to the NBS hypothesis, can explain the devastating effect of a Thr-->Ala mutation in a well-characterized resistance gene product. According to the new hypothesis, regions located N-terminal and C-terminal to the modeled portion, containing highly conserved sequence motifs, could form a separate domain.
Assuntos
Genes de Plantas , Doenças das Plantas , Sequência de Aminoácidos , Substituição de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The 6.7 murine monoclonal antibody (mAb) recognizes the human CD18 antigen and is therefore of interest as an anti-inflammatory agent. The 6.7 heavy variable chain (VH) was humanized using the closest human germline sequence as the template on to which to graft the murine complementary determining regions (CDRs). Two versions were proposed, one in which the residue proline 45 of the murine form was maintained and another in which this framework residue was changed to the leucine found in the human sequence. These VH humanized versions were expressed in the yeast Pichia pastoris as hemi-humanized single-chain Fv (scFvs), with the VL from the murine antibody. The scFv from the murine antibody was also expressed. The binding activities of the murine and both hemi-humanized scFvs were determined by flow cytometry analysis. All the constructions were able to recognize human lymphocytes harboring CD18, indicating successful humanization with transfer of the original binding capability. Some differences between the two hemi-humanized versions were observed. The method used was simple and straightforward, with no need for refined structural analyses and could be used for the humanization of other antibodies.
Assuntos
Antígenos CD18/imunologia , Região Variável de Imunoglobulina/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Pichia/genética , Homologia de Sequência de AminoácidosRESUMO
Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Five alpha-amylase inhibitors of the structural 0.19 family were isolated from wheat kernels, and assayed against three insect alpha-amylases and porcine pancreatic alpha-amylase, revealing several intriguing differences in inhibition profiles, even between proteins sharing sequence identity of up to 98%. Inhibition of the enzyme from a commercially important pest, the bean weevil Acanthoscelides obtectus, is observed for the first time. Using the crystal structure of an insect alpha-amylase in complex with a structurally related inhibitor, models were constructed and refined of insect and human alpha-amylases bound to 0.19 inhibitor. Four key questions posed by the differences in biochemical behaviour between the five inhibitors were successfully explained using these models. Residue size and charge, loop lengths, and the conformational effects of a Cys to Pro mutation, were among the factors responsible for observed differences in specificity. The improved structural understanding of the bases for the 0.19 structural family inhibitor specificity reported here may prove useful in the future for the rational design of inhibitors possessing altered inhibition characteristics.
Assuntos
Inibidores Enzimáticos/química , Triticum/química , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Insetos/efeitos dos fármacos , Insetos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Pâncreas/enzimologia , Controle de Pragas , Ligação Proteica , Conformação Proteica , Saliva/enzimologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , alfa-Amilases/químicaRESUMO
Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Secale/química , Animais , Cromatografia por Troca Iônica , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Inibidores da Tripsina/farmacologia , alfa-Amilases/antagonistas & inibidoresRESUMO
Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the first eukaryotic pyruvate kinase crystal structure in the T-state is reported. A comparison of the leishmania and yeast R-state enzymes reveals fewer differences than the previous comparison of Escherichia coli T-state and rabbit muscle non-allosteric enzymes. Structural changes related to the allosteric transition can therefore be distinguished from those that are a consequence of the inherent wide structural divergence between bacterial and mammalian proteins. The allosteric transition involves significant changes in a tightly packed array of eight alpha helices at the interface near the catalytic site. At the other interface the allosteric transition appears to be accompanied by the bending of a ten-stranded intersubunit beta sheet adjacent to the effector site. Helix Calpha1 makes contacts to the N-terminal helical domain and bridges both interfaces. A comparison of the effector sites of the leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops comprising residues 443-453 and 480-489 adopt very different conformations in the two enzymes, and Lys453 and His480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These differences offer an opportunity for the design of drugs that would bind to the trypanosomatid enzymes but not to those of the mammalian host.
Assuntos
Leishmania mexicana/enzimologia , Piruvato Quinase/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Cristalografia por Raios X , Leishmania mexicana/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genéticaRESUMO
The study of the plant oncogene rolA has been hampered by a lack of structural information. Here we show that, despite a lack of significant sequence similarity to proteins of known structure, the rolA sequence adopts a known fold; that of the papillomavirus E2 DNA-binding domain. This fold is reliably identified by modern threading programs, which consider predicted secondary structure, but not by others. Although the rolA sequence is only around 16% identical to those of the available template structures, a structural model could be built that performed well against protein structure verification programs. The adopted strategy involved alignment corrections, justified by multiple model building and evaluation, with particular attention paid to the hydrophobic core residues. We find that rolA protein is predicted to resemble the template proteins in two key aspects; existence as a dimer and ability to bind DNA. rolA protein has recently been shown experimentally to possess DNA binding ability. This model predicts Lys 24 and Arg 27 to be involved in sequence-specific interactions and eight other residues to hydrogen-bond phosphate groups of the DNA.