Abstract
We have previously shown that protein prenylation occurs in the Trypanosomatids Trypanosoma brucei (T. brucei), Trypanosoma cruzi, and Leishmania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect (procyclic) form T. brucei. A PFT that transfers the farnesyl group from farnesyl pyrophosphate to a cysteine that is 4 residues upstream of the C terminus of the Ras GTP-binding protein RAS1-CVIM has now been purified 60,000-fold to near homogeneity from procyclic T. brucei. By screening a mixture of hexapeptides SSCALX (X is 20 different amino acids), it was found that SSCALM binds to T. brucei PFT with sub-micromolar affinity, and affinity chromatography using this peptide was a key step in the purification of this enzyme. On SDS-polyacrylamide gel electrophoresis, the enzyme migrates as a pair of bands with apparent molecular masses of 61 and 65 kDa, and thus its subunits are approximately 30% larger than those of the mammalian homolog. The 61-kDa band was identified as the putative beta-subunit by photoaffinity labeling with a 32P-labeled analog of farnesyl pyrophosphate. Mimetics of the C-terminal tetrapeptide of prenyl acceptors have been previously shown to inhibit mammalian PFT, and these compounds also inhibit T. brucei PFT with affinities in the nanomolar to micromolar range, although the structure-activity relationship is very different for parasite versus mammalian enzyme. Unlike mammalian cells, the growth of bloodstream T. brucei is completely inhibited by low micromolar concentrations of two of the PFT inhibitors, and these compounds also block protein farnesylation in cultured parasites. These compounds also potently block the growth of the intracellular (amastigote) form of T. cruzi grown in fibroblast host cells. The results suggest that protein farnesylation is a target for the development of anti-trypanosomatid chemotherapeutics.
Highlights
We have previously shown that protein prenylation occurs in the Trypanosomatids Trypanosoma brucei (T. brucei), Trypanosoma cruzi, and Leishmania mexicana and that protein farnesyltransferase (PFT) activity can be detected in cytosolic extracts of insect form T. brucei
These results have been explained by the facts that in the presence of PFT inhibitors, K-Ras and N-Ras become geranylgeranylated instead of farnesylated in cells [30] and that geranylgeranylation of Ras proteins is sufficient for proliferation of normal and Ras-trans
Purification of T. brucei PFT—Our previous study of protein prenylation in T. brucei showed that PFT activity was detected in cytosol of the procyclic form [36]
Summary
A HETERODIMER OF 61- AND 65-kDa SUBUNITS AS A NEW TARGET FOR ANTIPARASITE THERAPEUTICS*. Recent studies have shown that protein prenylation occurs in protozoan parasites Giardia lamblia [34], Trypanosoma brucei [35, 36], Trypanosoma cruzi [37], Leishmania mexicana [37], and in Schistosoma mansoni [38] This is based on the observation that proteins in these parasites become radiolabeled when cells are cultured in the presence of radiolabeled mevalonic acid (MVL), the universal precursor of prenyl groups. None of these prenylated parasite proteins have been identified, but a major group includes those of molecular mass of ϳ25 kDa, which is typical of small GTP-binding proteins that belong to the Ras superfamily of regulatory proteins. These results suggest that trypanosomatid PFT is an ideal target for the development of chemotherapeutics
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