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1.
Nature ; 530(7589): 233-6, 2016 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-26863983

RESUMO

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the ß2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum ß2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this ß2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Desenho de Fármacos , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Animais , Antimaláricos/efeitos adversos , Antimaláricos/toxicidade , Artemisininas/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Sinergismo Farmacológico , Ativação Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Plasmodium/crescimento & desenvolvimento , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Inibidores de Proteassoma/efeitos adversos , Inibidores de Proteassoma/toxicidade , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Especificidade da Espécie , Especificidade por Substrato/efeitos dos fármacos
2.
Anal Biochem ; 468: 22-7, 2015 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-25281458

RESUMO

In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.


Assuntos
Calpaína/metabolismo , Plasmodium chabaudi/enzimologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Cálcio/metabolismo , Calpaína/análise , Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Leupeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Proteínas de Protozoários/análise , Proteínas de Protozoários/antagonistas & inibidores , Espectrometria de Fluorescência
3.
Parasitol Res ; 112(11): 3757-70, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23949311

RESUMO

Epigenetic reprogramming of host genes via DNA methylation is increasingly recognized as critical for the outcome of diverse infectious diseases, but information for malaria is not yet available. Here, we investigate the effect of blood-stage malaria of Plasmodium chabaudi on the DNA methylation status of host gene promoters on a genome-wide scale using methylated DNA immunoprecipitation and Nimblegen microarrays containing 2,000 bp oligonucleotide features that were split into -1,500 to -500 bp Ups promoters and -500 to +500 bp Cor promoters, relative to the transcription site, for evaluation of differential DNA methylation. Gene expression was analyzed by Agilent and Affymetrix microarray technology. Challenging of female C57BL/6 mice with 10(6) P. chabaudi-infected erythrocytes resulted in a self-healing outcome of infections with peak parasitemia on day 8 p.i. These infections induced organ-specific modifications of DNA methylation of gene promoters. Among the 17,354 features on Nimblegen arrays, only seven gene promoters were identified to be hypermethylated in the spleen, whereas the liver exhibited 109 hyper- and 67 hypomethylated promoters at peak parasitemia in comparison with non-infected mice. Among the identified genes with differentially methylated Cor-promoters, only the 7 genes Pigr, Ncf1, Klkb1, Emr1, Ndufb11, and Tlr6 in the liver and Apol6 in the spleen were detected to have significantly changed their expression. Remarkably, the Cor promoter of the toll-like receptor Tlr6 became hypomethylated and Tlr6 expression increased by 3.4-fold during infection. Concomitantly, the Ups promoter of the Tlr1 was hypermethylated, but Tlr1 expression also increased by 11.3-fold. TLR6 and TLR1 are known as auxillary receptors to form heterodimers with TLR2 in plasma membranes of macrophages, which recognize different pathogen-associated molecular patterns (PAMPs), as, e.g., intact 3-acyl and sn-2-lyso-acyl glycosylphosphatidylinositols of P. falciparum, respectively. Our data suggest therefore that malaria-induced epigenetic fine-tuning of Tlr6 and Tlr1 through DNA methylation of their gene promoters in the liver is critically important for initial recognition of PAMPs and, thus, for the final self-healing outcome of blood-stage infections with P. chabaudi malaria.


Assuntos
Interações Hospedeiro-Patógeno , Fígado/parasitologia , Plasmodium chabaudi/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Baço/parasitologia , Receptor 1 Toll-Like/genética , Receptor 6 Toll-Like/genética , Animais , Imunoprecipitação da Cromatina , Metilação de DNA , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Plasmodium chabaudi/enzimologia , Baço/patologia
4.
Exp Parasitol ; 135(1): 166-74, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23830988

RESUMO

Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Cisteína Proteases/imunologia , Vacinas Antimaláricas , Malária/prevenção & controle , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/imunologia , Animais , Anopheles/parasitologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Cisteína Proteases/análise , Cisteína Proteases/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Eritrócitos/parasitologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Plasmodium berghei/fisiologia , Plasmodium chabaudi/crescimento & desenvolvimento , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas
5.
Bioorg Med Chem Lett ; 22(16): 5174-6, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22818080

RESUMO

Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloroquine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activities. The synthesized derivatives showed CQ-resistance reversing effects. They also reacted with glutathione (GSH) both enzymatically and chemically, and inhibited glutathione reductase activity. 7-Benzyl derivative, which was obtained by reduction of the olefin group in α,ß-unsaturated ketone structure of BNTX, also exhibited CQ-resistance reversing effect, but its potency was significantly lower than that of BNTX. These outcomes suggested that the decrease in GSH level could be one of the mechanisms of CQ-resistance reversing effects induced by BNTX derivatives.


Assuntos
Antimaláricos/química , Compostos de Benzilideno/química , Resistência a Medicamentos/efeitos dos fármacos , Naltrexona/análogos & derivados , Plasmodium chabaudi/efeitos dos fármacos , Antimaláricos/síntese química , Antimaláricos/farmacologia , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/farmacologia , Cloroquina/farmacologia , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Naltrexona/síntese química , Naltrexona/química , Naltrexona/farmacologia , Plasmodium chabaudi/enzimologia
6.
Malar J ; 11: 156, 2012 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-22564457

RESUMO

BACKGROUND: The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored. RESULTS: In the present work, it was demonstrated by mass spectrometry analysis that Plasmodium parasites (Plasmodium chabaudi and Plasmodium falciparum) internalize and process plasma kininogen, thereby releasing vasoactive kinins (Lys-BK, BK and des-Arg9-BK) that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140. CONCLUSIONS: In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen.


Assuntos
Cisteína Endopeptidases/metabolismo , Cininogênios/metabolismo , Cininas/metabolismo , Plasmodium chabaudi/enzimologia , Plasmodium falciparum/enzimologia , Animais , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Cobaias , Humanos , Íleo/efeitos dos fármacos , Espectrometria de Massas , Contração Muscular/efeitos dos fármacos , Plasmodium chabaudi/metabolismo , Plasmodium falciparum/metabolismo , Proteólise
7.
J Pineal Res ; 50(1): 64-70, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20964707

RESUMO

Calcium (Ca(2+) ) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra-erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca(2+) . Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca(2+) and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca(2+) within the parasite, because buffering Ca(2+) changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca(2+) signals within the parasite, as did the application of a membrane-permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca(2+) and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca(2+) evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Melatonina/farmacologia , Plasmodium chabaudi/enzimologia , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Malária/parasitologia , Camundongos , Microscopia Confocal
8.
Int J Biol Macromol ; 45(4): 399-406, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19615402

RESUMO

Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization.


Assuntos
Modelos Moleculares , Plasmodium chabaudi/enzimologia , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Linhagem Celular Tumoral , Cristalografia por Raios X , Imunofluorescência , Genoma/genética , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/imunologia , Análise de Sequência de DNA
9.
Exp Parasitol ; 123(2): 122-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19538959

RESUMO

Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Glutationa/metabolismo , Inibidores da Protease de HIV/farmacologia , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Antirretrovirais/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Concentração Inibidora 50 , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Nelfinavir/farmacologia , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/metabolismo , Ritonavir/farmacologia , Saquinavir/farmacologia
10.
Exp Parasitol ; 122(2): 97-105, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19292986

RESUMO

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction. Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.


Assuntos
Anopheles/parasitologia , Cisteína Endopeptidases/análise , Plasmodium chabaudi/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Western Blotting , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Plasmodium chabaudi/genética , Plasmodium chabaudi/imunologia , Dobramento de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
11.
Mol Microbiol ; 65(1): 27-40, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17581118

RESUMO

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.


Assuntos
Resistência a Medicamentos/genética , Genes de Protozoários , Mutação , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/enzimologia , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Antimaláricos/farmacologia , Artemisininas/farmacologia , Artesunato , Cloroquina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos CBA , Modelos Moleculares , Testes de Sensibilidade Parasitária , Plasmodium chabaudi/genética , Sesquiterpenos/farmacologia
12.
Acta Trop ; 97(2): 212-8, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16329985

RESUMO

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin-the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Malária/parasitologia , Plasmodium chabaudi/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Compostos Cromogênicos/metabolismo , DNA de Protozoário/química , DNA de Protozoário/genética , Modelos Animais de Doenças , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Plasmodium chabaudi/genética , Plasmodium chabaudi/metabolismo , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de Proteína , Vacúolos/enzimologia
13.
Mol Biochem Parasitol ; 136(1): 43-50, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15138066

RESUMO

The genes encoding enzymes involved in glutathione (GSH) metabolism may modulate responses to antimalarial drugs, but the role of most of them in antimalarial drug resistance has scarcely been investigated. Using an in silico/PCR combined approach, we have isolated from Plasmodium chabaudi, full sequences of five Plasmodium falciparum gene orthologues involved in GSH metabolism: the gamma-glutamylcysteine synthetase (Pc-gammagcs), glutathione-synthetase (Pc-gs), glutathione peroxidase (Pc-gpx), glutathione reductase (Pc-gr) and glutathione-S-transferase (Pc-gst). DNA sequencing of these genes from drug sensitive parasites, P. chabaudi AS (0CQ), and ones isolated from parasite lines that show genetically stable resistance to chloroquine (CQ) at low, intermediate and high levels, AS (3CQ), AS (15CQ) and AS (30CQ), respectively, revealed no point mutations in the resistant parasites. We used these sequences to design internal oligonucleotide primers to compare relative mRNA amounts of these genes between all P. chabaudi clones, in untreated mice or following CQ treatment with sub-curative doses, by real-time PCR. Analysis of three independent experiments revealed that transcription levels of the Pc-gammagcs, Pc-gs, Pc-gpx, Pc-gr and Pc-gst genes were not changed between chloroquine sensitive and resistant parasite clones, and that treatment with chloroquine did not induce an alteration in the expression of these genes in sensitive or resistant parasites. We concluded that chloroquine resistance in this species is determined by a mechanism that is independent of these genes, and most likely, of GSH metabolism.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Glutationa/metabolismo , Plasmodium chabaudi/enzimologia , Animais , Antimaláricos/administração & dosagem , Cloroquina/administração & dosagem , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Malária/parasitologia , Camundongos , Dados de Sequência Molecular , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/genética
14.
Acta Trop ; 89(1): 1-12, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14636976

RESUMO

Intraerythrocytic malaria parasites degrade haemoglobin to provide nutrients for their own growth and maturation. Plasmodium aspartic proteases known as plasmepsins play an important role on haemoglobin degradation and are being studied as drug targets for chemotherapy of malaria. The rodent model for human malaria, Plasmodium chabaudi, is an experimentally good model for therapy drug design. The gene encoding an aspartic protease precursor (proplasmepsin) from the rodent malaria parasite P. chabaudi was cloned and sequenced. A theoretical 3D structure model was constructed by comparative homology and used for superimposition with other known models. Analysis of the P. chabaudi and Plasmodium yoelli genomes revealed in both the presence of at least seven plasmepsins and each one has sequence similarity to its plasmepsin counterpart of the human malaria Plasmodium falciparum. The predicted proteins were confirmed as plasmepsins by detection on Blocks Database of three characteristic blocks of the eukaryotic and viral aspartic protease family. Analysis of the proline-rich loop amino acid sequence of these plasmepsins suggests that they constitute characteristic motifs of each plasmepsin group suggesting that these sequence variations are related with different substrate specificities.


Assuntos
Ácido Aspártico Endopeptidases/genética , Modelos Moleculares , Plasmodium chabaudi/genética , Sequência de Aminoácidos , Animais , Genoma , Humanos , Camundongos , Dados de Sequência Molecular , Plasmodium chabaudi/enzimologia , Reação em Cadeia da Polimerase
15.
Braz J Med Biol Res ; 36(11): 1465-9, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14576900

RESUMO

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37 degrees C with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 microM - 75%), staurosporine (1 microM - 58%), R03 (1 microM - 75%), and tyrphostins B44 (100 microM - 66%) and B46 (100 microM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.


Assuntos
Inibidores Enzimáticos/farmacologia , Eritrócitos/parasitologia , Plasmodium chabaudi/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Ciclo Celular/efeitos dos fármacos , Feminino , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium chabaudi/enzimologia
17.
Parasitol Res ; 84(7): 552-8, 1998 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-9694371

RESUMO

The major leucine aminopeptidase of the rodent malarial parasite Plasmodium chabaudi chabaudi was partially purified using a combination of high-pressure liquid chromatography on a size-exclusion column and affinity chromatography using the aminopeptidase-specific inhibitor bestatin as the ligand. The purified enzyme showed simple Michaelis-Menten kinetics when the fluorogenic peptide analogue leucyl-7-amino-4-methyl-courmarin served as the substrate, and it was strongly inhibited by both bestatin (Ki = 50.7 +/- 21.0 nM) and nitrobestatin (Ki = 2.51 +/- 0.2 nM) in a competitive manner. These inhibitors were also potent blockers of the growth of P. c. chabaudi and the human parasite P. falciparum in culture, and nitrobestatin was again the more potent. Therefore, the leucine aminopeptidase represents an important target to which novel anti-malarial agents could be directed.


Assuntos
Antimaláricos/farmacologia , Leucina/análogos & derivados , Leucil Aminopeptidase/antagonistas & inibidores , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Leucina/farmacologia , Leucil Aminopeptidase/isolamento & purificação , Leucil Aminopeptidase/metabolismo , Camundongos , Plasmodium chabaudi/enzimologia , Plasmodium chabaudi/crescimento & desenvolvimento , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Inibidores de Proteases/farmacologia
19.
J Eukaryot Microbiol ; 41(2): 119-23, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8167617

RESUMO

Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately thermophilic. Each has an apparent molecular weight of 80,000 +/- 10,000, determined by high performance liquid chromatography on a calibrated SW500 column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho-phenanthroline and are thus metallo-aminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be Zn2+.


Assuntos
Aminopeptidases/isolamento & purificação , Plasmodium berghei/enzimologia , Plasmodium chabaudi/enzimologia , Plasmodium falciparum/enzimologia , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie
20.
Parasitology ; 107 ( Pt 1): 1-10, 1993 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8355992

RESUMO

Plasmodium falciparum, P. knowlesi and P. chabaudi showed a significant activity of methylenetetrahydrofolate reductase (MTHFR). The presence of this enzyme completes the methionine synthesis cycle, in which the one-carbon fragment from serine side-chain can be transferred to methionine. However, while metabolic labelling of methionine from L-3 [14C]serine could not be demonstrated in P. falciparum, the significance of MTHFR was implicated by a novel pathway for salvage of exogenous 5-methyltetrahydrofolate from the host cell. The methyl group of the cofactor was incorporated into methionine, and the folate cofactor was found in the same pool as that derived from de novo synthesis with p-aminobenzoic acid as the precursor, shown previously as polyglutamylated 5-methyltetrahydrofolate. It is proposed from these results that the function of MTHFR and the methionine synthesis cycle is not the supply of methionine, but the generation of active folate cofactors from more stable precursors salvaged by the parasites.


Assuntos
Eritrócitos/metabolismo , Metionina/biossíntese , Plasmodium falciparum/metabolismo , Tetra-Hidrofolatos/biossíntese , Tetra-Hidrofolatos/metabolismo , Animais , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2) , Modelos Biológicos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/análise , Plasmodium chabaudi/enzimologia , Plasmodium falciparum/enzimologia , Plasmodium knowlesi/enzimologia , Serina/metabolismo
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