Elizabeth A. Worthey

Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi

BackgroundThe trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids.ResultsBioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs.ConclusionTrypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules.

Evaluation of differential gene expression in Leishmania major Friedlin procyclics and metacyclics using DNA microarray analysis

The various Leishmania species are flagellated protozoans, responsible for a wide spectrum of human diseases. The sequence of the L. major genome is nearing completion and a large proportion of the identified genes have yet to be ascribed functions. DNA microarrays containing PCR-amplified DNA from a random amplified genomic library of L. major Friedlin (LmjF) [Mol. Biochem. Parasitol. 113 (2001) 337] were hybridized with fluorescent probes made from L. major Friedlin RNA from five time-points during differentiation from procyclics to metacyclics. The data were normalized for background and probe intensity and the relative abundance of RNA for each spot was calculated. Almost 15% (1387/9282) of the DNAs showed statistically significant (P<0.01) changes in expression (1.1-5-fold) during the transition, with 1.16% (108) showing up-regulation at two or more time-points and 0.14% (13) showing down-regulation. Northern blot analyses of selected genes confirmed these results. These studies confirmed the stage-specific expression of several known genes, as well as identifying a number of novel genes that are up-regulated in either procyclics or metacyclics.

Crystal structure of glyceraldehyde‐3‐phosphate dehydrogenase from Plasmodium falciparum at 2.25 Å resolution reveals intriguing extra electron density in the active site

The crystal structure of D‐glyceraldehyde‐3‐phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 Å resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD+ is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD+. This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials. Proteins 2006.

Importing statistical measures into Artemis enhances gene identification in the Leishmania genome project

BackgroundSeattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.ResultsHere we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.ConclusionAn improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily.

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well‐characterized N‐carbamoylsarcosine‐amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial‐associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter‐subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.

Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major.

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (Rfree = 0.229) at 1.60 A resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.

Structure of a ribulose 5‐phosphate 3‐epimerase from Plasmodium falciparum

The crystal structure of Pfal009167AAA, a putative ribulose 5‐phosphate 3‐epimerase (PfalRPE) from Plasmodium falciparum, has been determined to 2 Å resolution. RPE represents an exciting potential drug target for developing antimalarials because it is involved in the shikimate and the pentose phosphate pathways. The structure is a classic TIM‐barrel fold. A coordinated Zn ion and a bound sulfate ion in the active site of the enzyme allow for a greater understanding of the mechanism of action of this enzyme. This structure is solved in the framework of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium. Proteins 2006.