RESUMEN
Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.
Asunto(s)
Dinoprost/biosíntesis , Prostaglandina-Endoperóxido Sintasas/aislamiento & purificación , Prostaglandina-Endoperóxido Sintasas/metabolismo , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Extractos Celulares , Células Cultivadas , Clonación Molecular , Dinoprost/metabolismo , Dinoprostona/biosíntesis , Dinoprostona/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Cinética , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Prostaglandina D2/biosíntesis , Prostaglandina D2/metabolismo , Prostaglandina H2 , Prostaglandina-Endoperóxido Sintasas/química , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandinas H/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Especificidad por Sustrato , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.
Asunto(s)
Glicerol Quinasa/metabolismo , Proteínas Protozoarias , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Expresión Génica , Genes Protozoarios , Glicerol Quinasa/genética , Glicerol Quinasa/aislamiento & purificación , Humanos , Cinética , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genéticaRESUMEN
Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphospha-tase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Site-directed mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.