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Biological characterization of de-ubiquitylating enzymes (UBPs/UCHs) in Plasmodium spp as potential drug targets
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A malária ainda constitui um grande problema de saúde pública e a resistência aos antimaláricos ameaça todos os esforços efectuados com vista ao combate e controle desta doença. Existe uma grande necessidade de se identificar novos compostos de preferência que actuem em novos alvos terapêuticos. A via da ubiquitinação/proteosoma já foi identificada como um alvo terapêutico interessante. Mutações nas enzimas de des-ubiquitilação (DUBs) que catalizam a remoção da ubiquitina estão associadas ao desenvolvimento de doenças infecciosas e não infecciosas.
Neste projecto quatro DUBs foram identificadas no genoma do parasita Plasmodium falciparum e foram caracterizadas. A expressão dos genes que codificam estas enzimas ao longo do ciclo de vida do parasita na presença e ausência de fármaco foi efectuada por RT-PCR.Anticorpos policlonais obtidos a partir de ratinhos foram utilizados para a deteção da abundancia das proteínas ao longo do ciclo de vida do parasita. Utilizou-se ainda a tecnica de transfeção com o objectivo de criar uma linha knockout para determinar se estas proteínas são essências para o parasita. Proteínas recombinantes foram expressas em células de E.coli e actividade enzimática das mesmas foi testada usando um substrato específico para as DUBs. O inibidor das DUBs com actividade antimalarica, curcumina foi usado quer in vitro para testar a sua actividade sobre as proteínas recombinantes, mas também in vivo no modelo de malaria roedora de Plasmodium chabaudi em associação com cloroquina e artemisinina.Um ensiao de proteomica foi também usado para ver que proteínas estão alteradas em resposta ao tratamento com curcumina.
Os resultados demonstram que em P. falciparum os genes pfuch-l1, pfuch-l3, pfuch-l54 e pfubp-8 são diferencialmente expressos ao longo do ciclo de vida do parasita e as respectivas proteínas são mais abundantes no estadio de trofozoito e esquizonte. O tratamento dos parasitas com artemisinina, cloroquina, curcumina induziu um aumento temporário na expressão dos genes seguido de um declínio. Não foi possível obter uma linha parasitária knockout pfuch-l1 e pfuch-l3 viável. As proteínas recombinantes foram expressas com sucesso em células de E. coli excepto a Pfuch-l54. As Pfuch-l1, Pfuch-l3, Pfubp-8 demonstraram actividade enzimática e interagiram com o susbstrato Ub-AMC. Os IC50 da curcumina nas proteínas recombinantes foram: Pfuch-l1 15μM, Pfuch-l3 25.4μM, Pfubp-8 10μM e para a proteina recombinante humana USP2, 5μM. A Curcumina quando testada nas células HepG2 apresenta alguma toxicidade in vitro, mas não apresenta uma alta toxicicidade em ratinhos e quando utilizada em associação com a cloroquina apresenta um efeito de sinergismo.Enquanto a associação da curcumina com artemisinina o resultado é antagónico.Os ensaios de proteomica em culturas de P. falciparum tratadas com curcumina revelaram 10 proteinas que se encontraram alteradas em resposta ao tratamento. Estas proteínas estão envolvidas no metabolismo do sulfato, tradução e degradação de proteínas, ciclo celular e organização celular.
Em conclusão, este trabalho demonstra que estas enzimas são potenciais alvos terapeuticos, mas será necessário mais estudos moleculares, bioquímicos e farmacológicos para aumentar a selectividade dos inibidores das DUBs para as enzimas do parasita e minimizar os danos nas proteínas do hospedeiro humano.
Malaria continues to be a major public healthconcern. Drug resistance continues to threaten all efforts made to control the disease. Hence there is a race to identify new antimalarial drugs that act on newer targets, in order to minimize the spread of drug resistance. The ubiquitin/proteasome pathway has been idientified as a potential drug target. Mutations in de-ubiquitylating enzymes (DUBs),which catalyze the removal of ubiquitin,havebeen associated with the developmentof infectiousand non infectious diseases. In this project four DUBs namely pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8were identified in the Plasmodiumfalciparumgenome and were characterized. Theexpression profile of genes encoding DUBsthroughout the parasite ́s life cycle with and without drug treatment wascarried out by RT-PCR.Polyclonal antibodies raised in mice were used to detect protein abundance in different stages of the parasite ́s life cycle. An attempt was made to produce a DUB knockoutlineand determine whether they are essential for the parasite. Recombinant proteins were expressed in E.colicells and their de-ubiquitylating activity was tested usinga specific substrate for DUBs.The activity of curcumin (a Dub inhibitor)was evalutedinvitroon the recombinant proteinsand its antimalarialactivity was testedin association with chloroquine and artemisininin anin vivorodent malaria model,Plasmodium chabaudi. A proteomics approach was also used to determine what proteins were deregulated in response to curcumin treatment.The results show that P.falciparumgenes pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8are differentially expressed throughout the parasite ́s life cycle and those proteins are more abundant at the trophozoite and schizont stages of the parasite.Treatment of parasites with artemisinin, chloroquine, and curcumin induced a transientincrease in the expression of those genes,followed by a steadydecrease in the gene expressionpattern.No viable pfuch-l1and pfuch-l3gene knockout lines were obtained.Recombinant proteins were successfully expressed in E.colicellswith the exception of Pfuch-l54.Pfuch-l1, Pfuch-l3, Pfubp-8demonstrated de-ubiquitylating activity by cleaving the substrate Ub-AMC. In vitroIC50 of curcumin towards recombinant Pfuch-l1was 15μM, for recombinant Pfuch-l3was 25.4μM and forPfubp-8was 10μM and for human USP2 was 5μM. Curcumin displayed some toxicity to the HepG2 cell lines, but the in vivoantimalarial activityassays in the rodent model of malaria Plasmodium chabaudishowedthat curcumin is non toxic to miceand the association of curcumin with chloroquine displayedsynergism whereas theassociation of curcumin with artemisinin showed antagonism. The proteomics assay performed in P.falciparumcultures treated with curcuminrevealed10deregulatedproteins.The proteins identified were involved in sulfur metabolism, protein translation and degradation, cell cycleand cellular organization. In conclusion, the presentstudyshowed that P.falciparumDUBs are indeed potential drug targets. However further molecular, biochemicaland phamacological studies will be required in ordertoturn the inhibitors more specific towards the parasite ́s enzymes andminimise damage to the host ́s proteins.
Malaria continues to be a major public healthconcern. Drug resistance continues to threaten all efforts made to control the disease. Hence there is a race to identify new antimalarial drugs that act on newer targets, in order to minimize the spread of drug resistance. The ubiquitin/proteasome pathway has been idientified as a potential drug target. Mutations in de-ubiquitylating enzymes (DUBs),which catalyze the removal of ubiquitin,havebeen associated with the developmentof infectiousand non infectious diseases. In this project four DUBs namely pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8were identified in the Plasmodiumfalciparumgenome and were characterized. Theexpression profile of genes encoding DUBsthroughout the parasite ́s life cycle with and without drug treatment wascarried out by RT-PCR.Polyclonal antibodies raised in mice were used to detect protein abundance in different stages of the parasite ́s life cycle. An attempt was made to produce a DUB knockoutlineand determine whether they are essential for the parasite. Recombinant proteins were expressed in E.colicells and their de-ubiquitylating activity was tested usinga specific substrate for DUBs.The activity of curcumin (a Dub inhibitor)was evalutedinvitroon the recombinant proteinsand its antimalarialactivity was testedin association with chloroquine and artemisininin anin vivorodent malaria model,Plasmodium chabaudi. A proteomics approach was also used to determine what proteins were deregulated in response to curcumin treatment.The results show that P.falciparumgenes pfuch-l1, pfuch-l3, pfuch-l54and pfubp-8are differentially expressed throughout the parasite ́s life cycle and those proteins are more abundant at the trophozoite and schizont stages of the parasite.Treatment of parasites with artemisinin, chloroquine, and curcumin induced a transientincrease in the expression of those genes,followed by a steadydecrease in the gene expressionpattern.No viable pfuch-l1and pfuch-l3gene knockout lines were obtained.Recombinant proteins were successfully expressed in E.colicellswith the exception of Pfuch-l54.Pfuch-l1, Pfuch-l3, Pfubp-8demonstrated de-ubiquitylating activity by cleaving the substrate Ub-AMC. In vitroIC50 of curcumin towards recombinant Pfuch-l1was 15μM, for recombinant Pfuch-l3was 25.4μM and forPfubp-8was 10μM and for human USP2 was 5μM. Curcumin displayed some toxicity to the HepG2 cell lines, but the in vivoantimalarial activityassays in the rodent model of malaria Plasmodium chabaudishowedthat curcumin is non toxic to miceand the association of curcumin with chloroquine displayedsynergism whereas theassociation of curcumin with artemisinin showed antagonism. The proteomics assay performed in P.falciparumcultures treated with curcuminrevealed10deregulatedproteins.The proteins identified were involved in sulfur metabolism, protein translation and degradation, cell cycleand cellular organization. In conclusion, the presentstudyshowed that P.falciparumDUBs are indeed potential drug targets. However further molecular, biochemicaland phamacological studies will be required in ordertoturn the inhibitors more specific towards the parasite ́s enzymes andminimise damage to the host ́s proteins.
Descrição
Palavras-chave
Parasitologia Médica Malária Enzimas Terapêutica
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical