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- El-Sayed, Najib M., et al.
(författare)
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The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.
- 2005
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 309:5733, s. 409-15
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Tidskriftsartikel (refereegranskat)abstract
- Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
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| 3. |
- Ochaya, Stephen, et al.
(författare)
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Characterization of a Trypanosoma cruzi acetyltransferase : cellular location, activity and structure
- 2007
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 152:2, s. 123-131
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Tidskriftsartikel (refereegranskat)abstract
- Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.
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| 4. |
- Ochaya, Stephen
(författare)
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Comparative genomics and molecular characterization of N-alpha acetyltransferase in trypanosomes for drug target identification
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Trypanosoma cruzi and Trypanosoma brucei are flagellated protozoan parasites that cause Chagas disease and African trypanosomiasis in Latin American and African countries, respectively. Currently, over 8 million people are infected with T. cruzi and about 25 million more are at risk. About half a million people are affected by T. brucei. Trypanosome species share many peculiar biological and biochemical features, such as RNA editing. In contrast, they exhibit profound differences at the level of host-parasite interaction and disease pathology. Both parasites are transmitted to their host via different insect vectors. There are no available vaccines, and the current treatments have severe adverse effects. We were involved in sequencing the T. cruzi genome, an initiative launched by the WHO to increase our knowledge of the molecular basis of the parasite. The aim of this thesis was to participate in the sequencing and analysis of the T. cruzi genome, and use the data to investigate acetyltransferase enzymes, presumably linked to important metabolic pathways, as possible drug targets. In Papers I and II, we describe genome sequencing and analysis of two distinct T. cruzi strains. One of the selected strains, CL Brener, was found to be a genetic hybrid of two divergent strains; and it contains about 22 000 genes, encoded on 700 scaffolds with a total genome size of 110 Mb. About 50% of the genes are of unknown function, and lack homology to other sequenced eukaryotes. Large numbers of members of surface molecule gene families, such as trans-sialidase, mucin, mucin-associated protein, and GP63 were found. Comparative analyses revealed that TcI had a smaller genome by up to about 11 Mb. The genome size difference was linked to genes encoding surface molecules and to other repeats and repeated genes. Additionally, six reading frames present in TcVI were not detected in TcI. Genetic polymorphisms such as, indels, microsatellites and SNPs were identified and analyzed. Many genes were found to be under different selective pressures in T. cruzi, indicating differential evolutionary rates, signifying their importance to parasite biology. Within syntenic regions, the two genomes have the same gene complement. Identified features warrant sequencing of further T. cruzi strains, and findings from our studies offer opportunities for more targeted functional studies as well as tools for epidemiology. In the second part of this thesis, Papers III to V, a Trypanosoma cruzi acetyltransferase gene family, identified in the genome project, was chosen for functional characterization as a first step to evaluate its potential as drug target. Acetyltransferases are responsible for protein acetylation, where an acetyl molecule is transferred from acetyl-Coenzyme A to lysine residues in a protein sequence, N-epsilon acetylation, and to N-termini of proteins or stability, protein-protein interaction, localization to organelles and acts as degradation signals. The impact of this post-translational modification in parasite is not known. We have identified T. cruzi NatC and A, and show that they are expressed in the three life cycle stages (epimastigote, trypomastigote, and amastigote). The catalytic and auxiliary subunits form a complex in vivo. Additionally, they partially co-sediment with the ribosome and may have both co-translational and post-translational protein acetylation functions. In epimastigote, the catalytic subunit of T. cruzi NatA was localized both in the nuclear periphery and in cytoplasm, whereas NatC was predominantly assigned to the cytoplasm. The auxiliary subunit of NatA was mainly confined to the cytoplasm with cytoskeletal-like labelling, whereas NatC showed a punctate profile. Interestingly, the staining patterns of the different subunits analysed for NatA and NatC differ between the life cycle stages, which suggests differential regulation and expression. The native substrates for NatC and predicted NatA, are similar to those described in yeast and humans,suggesting evolutionary conserved functions. The proteins appear to acetylate a large number of proteins Nterminally, suggesting that manipulation of the enzymes may simultaneously affect many cellular functions and thereby could interfere with or abolish infection. Additionally, our data indicate that NatC and A, may have both Nalpha and N-epsilon acetylation potential. Collectively, the genome analyses presented here have provided more molecular insights into the parasite's biology, and have narrowed the gaps between scientific communities working on parasite research. The identification of Nats and native substrates has hopefully laid a solid foundation for future study of Nats, which could provide chemo- therapeutic targets for parasitic diseases.
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| 5. |
- Ochaya, Stephen O
(författare)
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Mapping the genome and characterization of an acetyltransferase of Trypanosoma cruzi
- 2006
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Trypanosoma cruzi (T.cruzi) is a flagellate protozoan parasite that causes Chagas' disease and is most common in Latin American countries. The disease currently infects over 13 million persons and about 100 million more are at risk. Despite basic research for many years, there is neither a vaccine nor useful drugs available to combat the disease. We were involved in sequencing the genome of this parasite, launched by WHO in order to increase knowledge on the molecular biology of the parasite and disease pathology. In the first part of this thesis, we tried to elucidate the molecular karyotype (actual numbers and sizes of chromosomes in a cell) of T.cruzi, which was an essential part of the genome project, newly published in Science together with two other kinetoplastid genomes. For the karyotyping study, three strains were used. CL-Brener (lineage IIe), chosen as a reference strain in the genome project and two T. cruzi lineage 1 strains, Sylvio X 10/7 and CAI/72 for comparative purposes. Since the T cruzi chromosomes do not condense during cell division, we separated the intact chromosomes by PFGE using three different running conditions. We hybridized 239 cDNA markers (probes) to the PFGE separated chromosomes. Using densitometry analysis - the total number of chromosomes for CL-Brener was estimated to be 55 and 57 in the two other strains. Size differences between homologous chromosomes, especially for CL-Brener are very high, up to 173%, and may be due to chromosome rearrangements. Forty markers distributed into 15 linkage groups, were found to identify specific chromosomes or chromosome pairs, hence showing conservation of gene order (synteny) between strains. This data provides valuable information for the finishing of the CL- Brener genome sequence. In the second part of this thesis, a Trypanosoma cruzi acetyltransferase (TcAT) protein that was annotated in our laboratory during the genome sequencing was characterized as a first step to evaluate its future potential as a drug target. Acetyltransferases (ATs) in general are enzymes that transfer acetyl-Coenzyme A (AcCo A) to the Nterminal of a protein or a peptide. In principle, acetylation is a posttranslational modification present in a majority of eukaryotes, altering the properties of proteins in different ways. The consequences of acetylation include effects on protein stability, protein-protein interaction and DNA binding. Our analysis of TcAT shows that the gene is single copy. The TcAT motifs match the GCN5-related acetyltransferase (GNAT) family. Orthologous proteins are present in the other two kinetoplastids (Trypanosoma brucei and Leishmania). The protein appears to be more closely related to N-terminal acetyltransferases (ATs) than to histone acetyltransferases (HATs). The native protein has a cytosolic location. It is expressed in the three different life-cycle stages of the parasite, determined by western blot. The recombinant protein has an autoacetylation activity. The 3D topology model prediction for TcAT is typical of the GNAT super family of ATs. The N-terminal of the human ortholog (HYPO-HUMAN) is elongated and there are three extra alpha helices predicted. Interestingly, kinetoplastids also have three extra helices but not in the same positions as in human. Some of the predicted functional sites where Acetyl CoA binds, and ligand binding sites were conserved in Kinetoplastids compared to other organisms including human. Proteins that interact with TcAT, native substrate specificity and metabolic pathway are not yet known. Despite that fact, the predicted protein structures of Kinetoplastids, amino acid, functional and ligand binding sites seem to differ from that of human counterpart and could be exploited and used in drug design against Trypanosomes and Leishmania.
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