RESUMO
A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in Escherichia coli. The automaturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally occurring form isolated from parasites. The S3-S3' subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. On the basis of the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 A structure of the PfPM2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 1-6, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Inibidores Enzimáticos/química , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/isolamento & purificação , Sítios de Ligação/genética , Catálise , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Malária Falciparum/enzimologia , Malária Falciparum/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Renaturação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Solubilidade , Relação Estrutura-Atividade , Especificidade por Substrato/genéticaRESUMO
We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved.
Assuntos
Lesões Encefálicas/diagnóstico , Lesões Encefálicas/metabolismo , Hipocampo/lesões , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroquímica/métodos , Sequência de Aminoácidos/fisiologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Lesões Encefálicas/fisiopatologia , Cromatografia Líquida/métodos , Creatina Quinase/análise , Creatina Quinase/metabolismo , Bases de Dados de Proteínas , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida/métodos , Hipocampo/fisiopatologia , Masculino , Espectrometria de Massas/métodos , Proteínas do Tecido Nervoso/análise , Neuroquímica/instrumentação , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/metabolismo , Valor Preditivo dos Testes , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrina/análise , Espectrina/metabolismo , Fatores de Tempo , Regulação para Cima/fisiologiaRESUMO
The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Plasmodium berghei/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Substituição de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Catálise , Domínio Catalítico , Ativação Enzimática , Expressão Gênica , Cinética , Plasmodium berghei/genética , Redobramento de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A fluorescent affinity tag (FAT) was synthesized and was utilized to selectively modify phosphorylated serine and threonine residues via beta-elimination and Michael addition chemistries in a 'one-step' reaction. This labeling technique was used for covalent modification of both phosphoproteins and phosphopeptides, allowing identification of these molecular species by fluorescence imaging after solution- or gel-based separation methods. In addition to the strong fluorescence of the rhodamine tag, a commercially available antibody can be used to enrich low-abundance post-labeled phosphopeptides present in complex mixtures. Application of this methodology to phosphorylation-site mapping has been evaluated for a phosphoprotein standard, bovine beta-casein. Initial results demonstrated low femtomole detection limits after fluorescence image analysis of FAT-labeled proteins or peptides.