The Order Kinetoplastida contains several pathogens of medical importance, including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. These organisms employ an unusual mechanism of gene expression that involves polycistronic transcription of protein-coding genes, with mature mRNA being generated via trans-splicing of a common 5'exon, the spliced leader, and addition of a poly(A) tail at the 3'end. To date there is no evidence for regulation of protein expression at the level of transcription initiation in kinetoplastids. In contrast, most of the regulation occurs post-transcriptionally. One level of control is the stability of the protein. The molecular determinants of protein stability, localization and turnover have not been characterized in kinetoplastids. It is important to understand protein dynamics in the kinetoplastid parasites because the loss and gain of specific proteins is key to fulfilling the transition between hosts in the parasite lifecycle. The hypothesis underlying this application is that the stability, function, and localization of a few hundred Trypanosoma brucei proteins will be modified by covalent attachment with the ubiquitin-like protein modifier SUMO.
The Aims of this proposal are: 1) To determine the identity of T. brucei proteins that are conjugated with His8-tagged SUMO by stringent affinity purification followed by an exquisitely sensitive variant of mass spectrometry referred to as 'MudPIT'. 2) Select targets of SUMO conjugation that are likely involved in transcription and/or RNA processing will be challenged and validated experimentally. Given the importance of post-transcriptional gene regulation in kinetoplastids coupled with the availability of MudPIT and the T. brucei genome database, this is a project that will yield results of interest to parasitologists and eukaryotic cell biologists alike.

Public Health Relevance

The causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease are transmitted from person to person via insect vectors. The pathogens'protein compositions are distinct in both insect and mammalian stages and needs to change rapidly during the transition between hosts. This proposal seeks to understand how SUMO contributes to the function, localization, and stability of proteins in the procyclic (insect) stage, with the long-term goal to understand the unique and critical features of this process that allow the parasite to progress through its lifecycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI079639-01A1
Application #
7659346
Study Section
Special Emphasis Panel (ZRG1-IDM-K (03))
Program Officer
Mcgugan, Glen C
Project Start
2009-05-05
Project End
2011-04-30
Budget Start
2009-05-05
Budget End
2010-04-30
Support Year
1
Fiscal Year
2009
Total Cost
$231,000
Indirect Cost
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095