RESUMO
Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.
Assuntos
Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismo , Sítios de Ligação , Catálise , Ativação Enzimática , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Humanos , Kinetoplastida/classificação , Kinetoplastida/enzimologia , Kinetoplastida/genética , Modelos Moleculares , Fosfoglicerato Quinase/genética , Filogenia , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis) and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors.
Assuntos
Antiprotozoários/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Kinetoplastida/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Antiprotozoários/química , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Parasita/efeitos dos fármacos , Humanos , Kinetoplastida/enzimologia , Kinetoplastida/fisiologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Nifurtimox/química , Nifurtimox/farmacologia , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologiaRESUMO
We report the characterization of cell-associated and extracellular peptidases of Bodo sp., a free-living flagellate of the Bodonidae family, order Kinetoplastida, which is considered ancestral to the trypanosomatids. This bodonid isolate is phylogenetically related to Bodo caudatus and Bodo curvifilus. The proteolytic activity profiles of Bodo sp. were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing co-polymerized gelatin, casein, hemoglobin, or bovine serum albumin as substrates. The enzymatic complex degraded gelatin better in acidic pH, and under these conditions four proteolytic bands (120, 100, 90, and 75 kDa) were detected in the cellular or extracellular extracts. Two peptidases (250 and 200 kDa) were exclusively detected with the substrate casein. All these enzymes belong to the serine peptidase class, based on inhibition by aprotinin and phenylmethylsulfonyl fluoride. This is the first biochemical characterization of peptidases in a free-living Bodo sp., potentially providing insight into the physiology of these protozoa and the evolutionary importance of peptidases to the order Kinetoplastida as some of these enzymes are important virulence factors in pathogenic trypanosomatids.
Assuntos
Kinetoplastida/enzimologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Serina Endopeptidases/análise , Serina Endopeptidases/genética , Animais , Aprotinina/farmacologia , Análise por Conglomerados , Cocos/parasitologia , DNA de Protozoário/química , DNA de Protozoário/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Eletroforese em Gel de Poliacrilamida/métodos , Inibidores Enzimáticos/farmacologia , Genes de RNAr , Dados de Sequência Molecular , Peso Molecular , Fluoreto de Fenilmetilsulfonil/farmacologia , Filogenia , Proteínas de Protozoários/química , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNA , Serina Endopeptidases/químicaRESUMO
The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi)-dependent PFKs, despite being ATP-dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica. It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity. PPi, even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site-directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs.