RESUMO
Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-ß-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.
Assuntos
Antimaláricos/farmacologia , Proteínas de Choque Térmico HSP72/metabolismo , Plasmodium falciparum/metabolismo , Alcaloides/farmacologia , Proteínas de Choque Térmico HSP72/efeitos dos fármacos , Concentração Inibidora 50 , Naftoquinonas/farmacologia , Plasmodium falciparum/efeitos dos fármacosRESUMO
Molecular chaperones have been used for the improved expression of target proteins within heterologous systems; however, the chaperone and target protein have seldom been matched in terms of origin. We have developed a heterologous co-expression system that allows independent expression of the plasmodial chaperone, PfHsp70, and a plasmodial target protein. In this study, the target was Plasmodium falciparum GTP cyclohydrolase I (PfGCHI), the first enzyme in the plasmodial folate pathway. The sequential expression of the molecular chaperone followed by the target protein increased the expression of soluble functional PfGCHI. His-tagged PfGCHI was successfully purified using nickel affinity chromatography, and the specific activity was determined by high performance liquid chromatography with spectrofluorometeric detection to be 5.93nmol/h/mg. This is the first report of a heterologous co-expression system in which a plasmodial chaperone is harnessed for the improved production and purification of a plasmodial target protein.
Assuntos
GTP Cicloidrolase/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Antimaláricos/uso terapêutico , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Escherichia coli , GTP Cicloidrolase/química , GTP Cicloidrolase/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Histidina/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Terapia de Alvo Molecular , Oligopeptídeos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de FluorescênciaRESUMO
The role of the TPR2B domain of Hop is as yet unknown. We have shown here by site directed mutagenesis and size exclusion chromatography for the first time that the TPR1 and TPR2B domains of Hop independently dimerized, and that the dimerization of TPR2B was not dependent on its predicted two-carboxylate clamp residues. Furthermore, our data indicated that the dimerization of Hop and its domains was not disrupted in the presence of Hsp70 and Hsp90 peptides.
Assuntos
Proteínas de Choque Térmico/química , Multimerização Proteica , Sequência de Aminoácidos , Dimerização , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas/isolamento & purificação , Alinhamento de SequênciaRESUMO
DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.
Assuntos
Adenosina Trifosfatases/metabolismo , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromatografia em Gel , Dimerização , Teste de Complementação Genética , Hidrólise , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de ProteínaRESUMO
Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) is a key enzyme in the synthesis of isoprenoids in the malaria parasite, using a pathway that is absent in the human host. This enzyme is receiving attention as it has been validated as a promising drug target. However, an impediment to the characterisation of this enzyme has been the inability to obtain sufficient quantities of the enzyme in a soluble and functional form. The expression of PfDXR from the codon harmonised coding region, under conditions of strongly controlled transcription and induction, resulted in a yield of 2-4 mg/L of enzyme, which is 8 to 10-fold higher than previously reported yields. The kinetic parameters Km, Vmax and kcat were determined for PfDXR using an NADPH-dependent assay. Residues K295 and K297, unique to species of Plasmodium and located in the catalytic hatch region; and residues V114 and N115, essential for NADPH binding, were mutated to resemble those found in E. coli DXR. Interestingly, these mutations decreased the substrate affinity of PfDXR to values resembling that of E. coli DXR. PfDXR-K295N, K297S and PfDXR-V114A, N115G demonstrated a decreased ability to turnover substrate by 4-fold and 2-fold respectively in comparison to PfDXR. This study indicates a difference in the role of the catalytic hatch in capturing substrate by species of Plasmodium. The results of this study could contribute to the development of inhibitors of PfDXR.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Chaperonas Moleculares/metabolismo , Pentosefosfatos/metabolismo , Plasmodium falciparum/enzimologia , Antimaláricos , Cinética , Plasmodium falciparum/química , Estrutura Secundária de ProteínaRESUMO
Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70-Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70-PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70-Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.
Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Resposta ao Choque Térmico , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Adenosina Trifosfatases/metabolismo , Citosol/metabolismo , Expressão Gênica , Hidrólise , Plasmodium falciparum/genética , Regulação para CimaRESUMO
A three-dimensional model of the malarial drug target protein PfDXR was generated, and validated using structure-checking programs and protein docking studies. Structural and functional features unique to PfDXR were identified using the model and comparative sequence analyses with apicomplexan and non-apicomplexan DXR proteins. Furthermore, we have used the model to develop an efficient approach to screen for potential tool compounds for use in the rational design of novel DXR inhibitors.