Characterisation of proline dehydrogenase and its contribution to energy metabolism in Trypanosoma brucei

Lamour, Nadia (2004) Characterisation of proline dehydrogenase and its contribution to energy metabolism in Trypanosoma brucei. PhD thesis, University of Glasgow.

Abstract

Both Leishmania major and Trypanosoma brucei undergo a complicated developmental cycle involving insect vectors and a mammalian host, subjecting these parasites to dramatic changes in their environment. It has been demonstrated that with some strains of T. brucei procyclic forms in culture, the amino acid proline is the favoured substrate for energy production. This correlates well with the normal environment of these forms in the tsetse fly, where free proline is an abundant source of energy. Proline utilisation requires two enzymes to convert proline to glutamate. In the first step, the oxidation of proline to pyrroline-5-carboxylate by proline dehydrogenase (PRODH) is coupled to the reduction of a cofactor, flavin adenine dinucleotide (FAD). In the second step, pyrroline-5-carboxylate is hydrolysed to give glutamic semialdehyde, which is oxidised by pyrroline-5-carboxylate dehydrogenase (P5CDH) to glutamate using the cofactor nicotinamide adenine dinucleotide (NAD+). Proline dehydrogenase genes have been identified in T. brucei and L. major. Both genes show significant homology to other eukaryotic proline dehydrogenase genes. Well conserved motifs, considered essential for the activity of the enzyme, are found in the predicted T. brucei and L. major protein sequences. As in other eukaryotes, the genes for the two enzymes (PRODH and P5CDH) involved in proline degradation are found in two different genomic locations, and encode two distinct polypeptides. In prokaryotes a single gene encodes both activities. A mitochondrial targeting motif present on the trypanosomatid enzymes, suggests that the mitochondrion is the subcellular localisation of this enzyme. This study went on to analyse the role of proline dehydrogenase in the energy metabolism of T. brucei procyclic forms. Growth studies using defined media revealed that procyclic forms in vitro can use either proline or glucose as an energy source. Interestingly, proline dehydrogenase activity is repressed in cells exposed to abundant glucose and, under these conditions, proline transport is also lower than in trypanosomes grown in low glucose concentrations. RNA interference was used to ablate proline dehydrogenase activity. These RNA interference experiments confirmed that the gene does encode the trypanosomal PRODH. Cells lacking this enzyme grew well in the presence of glucose but were unable to use proline as an energy source. Moreover, proline transport in this line also differed from that in wild type parasites. Proline could not be replaced by any other amino acid as an energy source in trypanosomes deprived of glucose. Even glutamate, which is an intermediate in the proline catabolic pathway, did not support growth.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Molecular biology, parasitology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Barrett, Dr. M. and Coombs, Prof. G.
• Date of Award: 2004
• Depositing User: Enlighten Team
• Unique ID: glathesis:2004-71235
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 08 Jun 2021 13:39
• URI: https://theses.gla.ac.uk/id/eprint/71235