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No description available.

Although F1-ATPase is extremely conserved among organisms, a putative subunit p18 was identified in Trypanosoma brucei F1-ATPase complex. To explore its function in the procylic, bloodstream and dyskinetoplastic trypanosomes, three different RNAi cell lines were created. Upon p18 silencing the F1-moiety structural integrity was impaired suggesting that p18 is indeed a bona fide subunit of this complex. Since F1-ATPase is crucial for the bloodstream form survival, its potential inhibitor from the 4-oxopiperidine-3,5-dicarboxylates class (JK-11) was examined. JK-11 inhibited growth of the bloodstream trypanosomes, decreased mitochondrial membrane potential and reduced ATPase and ATP synthase activity in mitochondrial lysates. Our results suggest that JK-11 may act on FoF1-ATP synthase/ATPase and its inhibition may contribute to the cytotoxicity of this drug.

In the mammalian bloodstream, the protozoan parasite Trypanosoma brucei undergoes differentiation from proliferative slender forms to arrested, transmissible, stumpy forms. This transition is associated with extensive cytological and metabolic changes that promote survival in the tsetse midgut, and also influences infection dynamics within the mammalian host. A number of genes involved in this transformation were recently identified using an RNAi library screen for resistance to pCPTcAMP, a membrane-permeable cyclic AMP analogue that induces differentiation. These molecules, referred to here as posST (positive mediators of STumpy formation), were thereafter validated to regulate the slender to stumpy transition, with many of them apparently comprising part of a signal transduction and effector pathway. However, it is unknown how these proteins act in relation to one another or are ordered within the pathway. To this end, null mutants were created for several posST components in differentiation-competent pleomorphic trypanosomes and, in this genetic background, other members of the predicted pathway expressed to test their ability to restore stumpy formation. Analysis of distinct combinations has been used to build a preliminary pathway structure model for the signalling events underlying trypanosome quorum sensing. In addition, phosphoproteomic analysis of two null mutants has revealed downstream signalling effects of two posST kinases, MEKK1 and YAK. A similar extragenic suppression approach was also applied to explore the interaction between the identified drivers of stumpy formation and the target of rapamycin kinase, TOR4, which has previously been shown to act as a negative regulator of stumpy formation in monomorphs. Dual ablation of TOR4 and posST components revealed insight into the intersection of stumpy-promoting and stumpy-inhibiting pathways. Finally, a chemical-genetic approach was used to investigate the posST pathway using two differentiation-inducing compounds: the previously studied E667, and GKI7, newly identified from a kinase inhibitor set. RNAi lines for different posST components were tested for their ability to undergo development in the presence of these compounds. An RNAi library screen using GKI7 identified putative new mediators of stumpy formation.

O Complexo de pré-replicação em T.brucei é composto por Orc1/Cdc6 e as helicases MCMs. Em um trabalho anterior mostramos que TbOrc1/Cdc6 pode ligar e hidrolisar ATP in vitro. Neste sentido, o objetivo deste trabalho é avaliar a importância da hidrólise e ligação de ATP para a formação e estabilidade do complexo pré-replicação de T.brucei. Para tanto, foram geradas proteínas recombinantes Orc1/Cdc6 de T. brucei mutadas nas regiões Walker A (TbOrc1/Cdc6K79T) ou sensor 2 (TbOrc1/Cdc6R251,252E) incapazes de ligar ou hidrolisar ATP, respectivamente. Finalmente, as células expressando TbOrc1/Cdc6K79T ou TbOrc1/Cdc6R251,252E foram avaliadas quanto a (i ) estabilidade da interação Orc1/Cdc6 -DNA, (ii) capacidade de estabilizar MCM no DNA, (iii) capacidade de replicar seu DNA. A mutação na região sensor 2 de T.brucei (TbOrc1/Cdc6R251,252E) reduziu drasticamente a atividade de ATPase em comparação com a proteína selvagem . TbOrc1/Cdc6 mutado no sitio de ligação ao ATP perdeu a capacidade de interagir com o ATP (TbOrc1/Cdc6K79T). A super expressão desses genes inibiu de forma significativa a proliferação celular, causou ineficiência no carregamento de MCM para o DNA e ocasionou falhas na progressão do ciclo celular, atrasando a fase S. / The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S.

O Complexo de pré-replicação em T.brucei é composto por Orc1/Cdc6 e as helicases MCMs. Em um trabalho anterior mostramos que TbOrc1/Cdc6 pode ligar e hidrolisar ATP in vitro. Neste sentido, o objetivo deste trabalho é avaliar a importância da hidrólise e ligação de ATP para a formação e estabilidade do complexo pré-replicação de T.brucei. Para tanto, foram geradas proteínas recombinantes Orc1/Cdc6 de T. brucei mutadas nas regiões Walker A (TbOrc1/Cdc6K79T) ou sensor 2 (TbOrc1/Cdc6R251,252E) incapazes de ligar ou hidrolisar ATP, respectivamente. Finalmente, as células expressando TbOrc1/Cdc6K79T ou TbOrc1/Cdc6R251,252E foram avaliadas quanto a (i ) estabilidade da interação Orc1/Cdc6 -DNA, (ii) capacidade de estabilizar MCM no DNA, (iii) capacidade de replicar seu DNA. A mutação na região sensor 2 de T.brucei (TbOrc1/Cdc6R251,252E) reduziu drasticamente a atividade de ATPase em comparação com a proteína selvagem . TbOrc1/Cdc6 mutado no sitio de ligação ao ATP perdeu a capacidade de interagir com o ATP (TbOrc1/Cdc6K79T). A super expressão desses genes inibiu de forma significativa a proliferação celular, causou ineficiência no carregamento de MCM para o DNA e ocasionou falhas na progressão do ciclo celular, atrasando a fase S. / The pre-replication complex in T.brucei is composed of at Orc1/Cdc6 and MCMs helicases. In a previous paper we showed that TbOrc1/Cdc6 can bind and hydrolyze ATP in vitro. Based on that, the objective of this study is to evaluate the importance of ATP binding and hydrolysis to the formation and stability of the pre - replication complex in T.brucei. For this purpose, T. brucei Orc1/Cdc6 recombinant proteins were generated mutated at regions on Walker A (TbOrc1/Cdc6K79T) and sensor 2 (TbOrc1/Cdc6R251 , 252E) in order to unable the ATP binding and hydrolyzation respectively . Finally , cells expressing TbOrc1/Cdc6K79T or TbOrc1/Cdc6R251 , 252E were evaluated for (i) stability of Orc1/Cdc6 - DNA interaction , (ii) ability to stabilize MCM in DNA , (iii) ability to replicate its DNA . The mutation in the sensor 2 region of T.brucei (TbOrc1/Cdc6R251 , 252E) drastically reduced the ATPase activity compared to the wild-type protein. TbOrc1/Cdc6 mutated in the ATP binding site has lost the ability to interact with ATP (TbOrc1/Cdc6K79T). The overexpression of these genes significantly inhibited cell proliferation causing inefficient loading of MCM DNA and led to failure in cell cycle progression by delaying the phase S.

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.