African trypansomes are parasitic protozoa that cause sleeping sickness in humans and nagana among domestic livestocks. We have been pursuing the mechanisms of developmental regulations in one of the species Trypanosoma brucei brucei, and have made some interesting findings in our recent studies. We: (1) identified and analyzed the C-terminal targeting signals in T. brucei glycosomal protein import; (2) identified and characterized an interferon (IFN)-gamma inducible MAP kinase homolog in bloodstream T. brucei; (3) identified, isolated and characterized the 20S and the activated 20S proteasome but indicated the apparent absence of 26 proteasome from T. brucei; (4) demonstrated that T. brucei differentiation from bloodstream into procyclic form may be initiated and completed within any particular phase of cell cycle without crossing any phase boundary; (5) demonstrated that a cysteine protease(s) may remove the variant surface glycoprotein from T. brucei during differentiation; (6) established the feasibility of complementing T. brucei mutants for direct gene clonings; 7) demonstrated the potential in developing the omithine decarboxylase-deficient mutants of T. brucei into vaccines against trypanosomiasis. For the future research plan, we will focus on two unique features in T. brucei, which have not yet found a parallel in other organisms, the process of differentiation from the bloodstream to the procyclic form and structures and functions of the 20S proteasomes. We propose: (1) to verify whether the T. brucei differentiation represents a simple, reversible change in pattern of gene expressions as a response to changes in environmental conditions. Many of the transcripts synthesized in the early phase of T. brucei differentiation have been cloned in the form of cDNA by suppression subtractive hybridizations. Each of the hundreds of cDNA's thus cloned will be analyzed for a profile of the genes turned on during the initial phase of T. brucei differentiation; (2) to identify and characterize the cysteine protease(s) apparently responsible in shedding the variant surface glycoprotein during T. brucei differentiation; 3) to analyze by mass spectrometry the structure of each subunit protein in T. brucei 20S proteasome and the proteasome activator protein PA26. The sequence data on the individual proteins thus obtained will be used to isolate the individual encoding genes or cDNA's, and the recombinant proteins used for the reconstitution of the proteasome. Inducibility of the activator protein PA26 and potential structural alterations of the 20S proteasome by IFNgamma treatment of T. brucei will be closely followed as they may provide a clue to regulated protein turnovers in T. brucei. Eventually, the potential alterations an the roles of 20S proteasomes played during T. brucei differentiation will be scrutinized. Information thus obtained may qualify T. brucei as a model for protein turnover studies bridging the gap between prokaryotic and advanced eukaryotic worlds.
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