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Prenylation of proteins in Trypanosoma brucei
K Yokoyama1, Y Lin, K D Stuart
1Department of Chemistry, University of Washington, Seattle 98195-1700, USA.
Abstract:
Prenyl modification of proteins by farnesyl and geranylgeranyl isoprenoids occurs in a variety of eukaryotic cells. Culturing of Trypanosoma brucei in the presence of [3H]mevalonolactone (which is hydrolyzed in cells to give mevalonic acid, the precursor of protein prenyl groups) and an inhibitor of mevalonic acid biosynthesis leads to the radiolabeling of a specific set of proteins when analyzed by gel electrophoresis. T. brucei proteins were also labeled when cells were cultured in the presence of [3H]farnesol or [3H]geranylgeraniol, and each prenol labels a distinct set of proteins. Unlike mammalian cells, only a few T. brucei proteins of molecular weights similar to those of the mammalian Ras superfamily of GTPase (20-30 kDa) were labeled with [3H]farnesol or [3H]geranylgeraniol. When the 0-55% ammonium sulfate fraction of T. brucei cytosol was fractionated on anion exchange chromatography, protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PGGT-I) activities were detected and elute as two distinct peaks. Partially purified T. brucei PFT and PGGT-I display partly different specificities toward prenyl acceptor substrates from those of mammalian protein prenyltransferases. As shown previously, rat PFT utilizes proteins ending in CVLS and CVIM as efficient prenyl acceptors and rat PGGT-I utilizes proteins ending in CVLL and CVIM in vitro. On the contrary, T. brucei PFT farnesylates a protein ending in CVIM but not CVLS or CVLL, and T. brucei PGGT-I preferentially geranylgeranylates a protein ending in CVLL.
Insights
This study investigates protein prenylation in Trypanosoma brucei, revealing distinct prenyltransferase specificities. T. brucei enzymes show unique substrate preferences compared to mammalian counterparts, impacting protein modification.
Area of Science:
- Biochemistry
- Molecular Biology
- Parasitology
Background:
- Prenyl modification is crucial for eukaryotic protein function.
- Trypanosoma brucei, a parasitic protozoan, undergoes protein prenylation.
- Understanding T. brucei prenylation is vital for drug development.
Purpose of the Study:
- To characterize protein prenyltransferases in T. brucei.
- To compare T. brucei prenyltransferase substrate specificity with mammalian enzymes.
- To identify potential drug targets for parasitic diseases.
Main Methods:
- Radiolabeling of T. brucei proteins using [3H]mevalonolactone, [3H]farnesol, and [3H]geranylgeraniol.
- Analysis of labeled proteins by gel electrophoresis.
- Fractionation of T. brucei cytosol to isolate protein farnesyltransferase (PFT) and protein geranylgeranyltransferase-I (PGGT-I) activities.
- In vitro assays to determine enzyme specificities.
Main Results:
- T. brucei prenylation involves distinct sets of proteins labeled by farnesol and geranylgeraniol.
- Unlike mammalian cells, few T. brucei proteins of Ras superfamily size were prenylated.
- Partially purified T. brucei PFT and PGGT-I exhibited different substrate specificities compared to rat enzymes.
- T. brucei PFT farnesylated proteins ending in CVIM, while PGGT-I preferentially geranylgeranylated proteins ending in CVLL.
Conclusions:
- T. brucei possesses unique protein farnesyltransferase and protein geranylgeranyltransferase-I enzymes.
- These enzymes display distinct substrate specificities compared to their mammalian homologs.
- The unique prenylation machinery of T. brucei represents a potential target for antiparasitic drug development.