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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021786-17
Application #
6488687
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Rogers, Martin J
Project Start
1984-12-01
Project End
2003-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
17
Fiscal Year
2002
Total Cost
$297,276
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Hu, Huiqing; Gourguechon, Stéphane; Wang, Ching C et al. (2016) The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31. J Biol Chem 291:15527-39
Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L et al. (2013) A minimal anaphase promoting complex/cyclosome (APC/C) in Trypanosoma brucei. PLoS One 8:e59258
Garlapati, Srinivas; Saraiya, Ashesh A; Wang, Ching C (2011) A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus. PLoS One 6:e18263
Sun, Lu; Wang, Ching C (2011) The structural basis of localizing polo-like kinase to the flagellum attachment zone in Trypanosoma brucei. PLoS One 6:e27303
Li, Zhi; Umeyama, Takashi; Li, Ziyin et al. (2010) Polo-like kinase guides cytokinesis in Trypanosoma brucei through an indirect means. Eukaryot Cell 9:705-16
Li, Ziyin; Umeyama, Takashi; Wang, C C (2009) The Aurora Kinase in Trypanosoma brucei plays distinctive roles in metaphase-anaphase transition and cytokinetic initiation. PLoS Pathog 5:e1000575
Gourguechon, Stephane; Wang, Ching C (2009) CRK9 contributes to regulation of mitosis and cytokinesis in the procyclic form of Trypanosoma brucei. BMC Cell Biol 10:68
Li, Ziyin; Lee, Ju Huck; Chu, Feixia et al. (2008) Identification of a novel chromosomal passenger complex and its unique localization during cytokinesis in Trypanosoma brucei. PLoS One 3:e2354
Li, Ziyin; Umeyama, Takashi; Wang, Ching C (2008) The chromosomal passenger complex and a mitotic kinesin interact with the Tousled-like kinase in trypanosomes to regulate mitosis and cytokinesis. PLoS One 3:e3814
Li, Ziyin; Lindsay, Megan E; Motyka, Shawn A et al. (2008) Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication. PLoS Pathog 4:e1000048

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