Glycosomes, the membrane-bound microbody organelles in the bloodstream form of Trypanosoma brucei, contain seven glycolytic enzymes and two glycerol metabolizing enzymes. Glycolysis is carried out inside the organelle at a high rate constituting the sole energy source for the organism. During its differentiation into the procyclic (insect) form, most of the enzymes in the glycosome are reduced to very low levels and replaced by phosphoenolpyruvate carboxykinase and malate dehydrogenase. We have been interested in the mechanism of protein import into the glycosome as well as the chain of events leading to the transformation of glycosomes during T. brucei differentiation. By transforming T. brucei with the firefly luciferase gene, we observed the import of luciferase into glycosomes in vivo. This import is dependent on the C- terminal tripeptide-ser-lys-leu-COOH of luciferase, a truncation of this tripeptide from luciferase keeps the latter in the cytoplasm of T brucei. Currently, we are performing site-directed mutagenesis of the three codons encoding this tripeptide in order to pinpoint the range of tripeptides capable of functioning as targeting signals for glycosomal protein import. The truncated luciferase will also serve as a reporter protein to be conjugated with a variety of peptides suspected of being the targeting signals in the past studies, and tested in the in vivo assay of glycosomal import. Analogs of these unique signal peptides may prove to be useful anti-trypanosomal agents. The differentiation of T. brucei is triggered by a temperature shift from 37oC to 26oC, which results in an immediate, albeit transient, decrease in protein synthesis in T. brucei. We plan to search for the potential inhibitor(s) of protein synthesis by an in vitro translation assay derived from T. brucei. We will also rely on a """"""""promoter trapping"""""""" technique to identify the genes expressed at the beginning of T. brucei differentiation. By incorporating a promoterless lacZ gene into a retroposon-like element TRS1 in T. brucei, one can transform T. brucei with the DNA construct and select for the transformants expressing the lacZ gene at the beginning of differentiation. The flanking regions of the lacZ gene will then reveal the real genes expressed at the crucial moment. We have also found that differentiation of the long-slender bloodstream form into the procyclic form of T. brucei does not require an intermediary or short- stumpy stage. This observation has ruled out any key role by ornithine decarboxylase (ODC) during the differentiation of T. brucei as originally anticipated. However, being the target for a new antitrypanosomal agent alpha-DL-difluoromethylornithine (DFMO), we have identified, cloned, sequenced and expressed the T. brucei ODC gene, and found it missing the C-terminal 36 amino acid peptide of mouse ODC. This discrepancy makes the T. brucei ODC remarkably stable inside eukaryotic cells, whereas the mouse ODC has an exceedingly short half-life of 20-30 minutes. This difference in enzyme stabilities may explain the in vivo selective action of DFMO against African trypanosomes. When mouse ODC was expressed inside T. brucei, the latter was also found incapable of degrading the mouse ODC. For our future research plan, we intend to crystallize T. brucei ODC and examine its three dimensional structure by X-ray diffraction analysis. The mechanism of inactivation of T. brucei ODC by DFMO will be studied by monitoring changes in absorption spectrum of the enzyme and release of fluoride ion from DFMO during the enzyme inactivation. The nucleophiles in the active pocket forming a Schiff's base with pyridoxal phosphate and covalent link with DFMO will be identified. Mouse ODC will be studied in a similar manner for potential discrepancies between the two enzymes.
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