Similar to other eukaryotes, the majority of mitochondrial proteins in Trypanosoma brucei, the infectious agent of a fatal disease (African trypanosomiasis) in human and domestic animals, are nuclear-encoded and need to be imported into mitochondria. However, the protein import machinery remains elusive in this organism. Fungi and higher eukaryotes possess three homologous proteins Tim17, Tim23, and Tim22, which formed two translocases (TIMs) in the inner membranes of mitochondria that have distinct specificities for different types of signal bearing proteins. In contrast, T. brucei possesses a single homolog of these proteins, TbTim17, which we found is present in a larger molecular mass protein complex in the mitochondrial inner membrane and is required for the import of both N-terminal and internal targeting signal containing proteins into mitochondria. Thus T. brucei may possess a single TIM complex capable of importing multiple types of matrix and inner membrane proteins. How a single protein in T. brucei performs functions of three different proteins in other eukaryotes remains enigmatic. We also have identified a Tim17- interacting protein (TbTim50) and genome searches indicate that there are other Tim50 related proteins in T. brucei. All of these variants possess a characteristic phosphatase domain. In addition to its role in mitochondrial protein import and cell growth, we found that TbTim50 over expression reduces mitochondrial ATP production via regulation of the expression level of the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. Together, this suggests that mitochondrial protein importer molecules such as Tim50 may be linked to other activities of this organelle besides importing proteins into mitochondrial matrix and inner membrane. In this project we will characterize the function of Tim50 and Tim50-related proteins in T. brucei and identify the Tim17 and Tim50 interacting domain(s) crucial for their function. We will also investigate how Tim50 is connected to VDAC expression, which is connected to regulation of mitochondrial metabolic activities. We will use various gene manipulation techniques, in vitro and in vivo protein import assays, FRET analysis, yeast two-hybrid analysis, and expression of TbTim50 mutants to study the function and protein/protein interactions of Tim50 and Tim17 in T. brucei. Characterization of the novel structure and function of T. brucei Tims may provide insights that can be utilized to define a new chemotherapeutic target for African trypanosomiasis.

Public Health Relevance

African trypanosomiasis, a fatal disease in humans as well as in domestic animals, is caused by the parasitic protozoa, Trypanosoma brucei. As available drugs for this disease are inadequate, it is critical to identify targets to design new drugs. Import of essential mitochondrial proteins is crucial for survival of this parasite in mammalian hosts. Therefore the unique structure and function of mitochondrial protein import molecules could be exploited as novel drug target(s).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Enhancement Award (SC1)
Project #
5SC1GM081146-06
Application #
8325571
Study Section
Special Emphasis Panel (ZGM1-MBRS-2 (GC))
Program Officer
Ainsztein, Alexandra M
Project Start
2007-08-01
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
6
Fiscal Year
2012
Total Cost
$327,375
Indirect Cost
$102,375
Name
Meharry Medical College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041438185
City
Nashville
State
TN
Country
United States
Zip Code
37208
Singha, Ujjal K; Hamilton, VaNae; Chaudhuri, Minu (2015) Tim62, a Novel Mitochondrial Protein in Trypanosoma brucei, Is Essential for Assembly and Stability of the TbTim17 Protein Complex. J Biol Chem 290:23226-39
Leaver, David J; Patkar, Presheet; Singha, Ujjal K et al. (2015) Fluorinated Sterols Are Suicide Inhibitors of Ergosterol Biosynthesis and Growth in Trypanosoma brucei. Chem Biol 22:1374-83
Weems, Ebony; Singha, Ujjal K; Hamilton, VaNae et al. (2015) Functional complementation analyses reveal that the single PRAT family protein of trypanosoma brucei is a divergent homolog of Tim17 in saccharomyces cerevisiae. Eukaryot Cell 14:286-96
Haubrich, Brad A; Singha, Ujjal K; Miller, Matthew B et al. (2015) Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth. J Lipid Res 56:331-41
Fullerton, Marjorie; Singha, Ujjal K; Duncan, Melanie et al. (2015) Down regulation of Tim50 in Trypanosoma brucei increases tolerance to oxidative stress. Mol Biochem Parasitol 199:9-18
Hamilton, Vanae; Singha, Ujjal K; Smith, Joseph T et al. (2014) Trypanosome alternative oxidase possesses both an N-terminal and internal mitochondrial targeting signal. Eukaryot Cell 13:539-47
Duncan, Melanie R; Fullerton, Marjorie; Chaudhuri, Minu (2013) Tim50 in Trypanosoma brucei possesses a dual specificity phosphatase activity and is critical for mitochondrial protein import. J Biol Chem 288:3184-97
Nes, Craigen R; Singha, Ujjal K; Liu, Jialin et al. (2012) Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms. Biochem J 443:267-77
Singha, Ujjal K; Hamilton, Vanae; Duncan, Melanie R et al. (2012) Protein translocase of mitochondrial inner membrane in Trypanosoma brucei. J Biol Chem 287:14480-93
Hall 3rd, Mack; Misra, Smita; Chaudhuri, Minu et al. (2011) Peptide aptamer mimicking RAD51-binding domain of BRCA2 inhibits DNA damage repair and survival in Trypanosoma brucei. Microb Pathog 50:252-62

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