Poster abstracts
Poster number 81 submitted by Kaitlin Klotz
Investigation of telomerase reverse transcriptase (TERT) ubiquitination and telomerase regulation in the parasitic protozoan, Trypanosoma brucei
Kaitlin Klotz (Department of Biological Science; University of North Carolina at Charlotte), Justin Davis (Department of Biological Sciences; University of North Carolina at Charlotte), Tiffany Barwell (Department of Biological Sciences; University of North Carolina at Charlotte), Nate Tomaszycki (Department of Biological Sciences; University of North Carolina at Charlotte), Bibo Li (Center for Gene Regulation in Health and Disease GRHD; Cleveland State University), Kausik Chakrabarti (Department of Biological Sciences; University of North Carolina at Charlotte)
Abstract:
Telomerase is a ribonucleoprotein complex that creates and extends caps of repetitive, non-coding DNA at the chromosome ends. It is minimally composed of a telomerase RNA (TR) which is the template for making repeats and a telomerase reverse transcriptase (TERT) which catalyzes the addition of telomeric repeats. The TR and TERT must interact to achieve telomerase activity. The interaction between TR and TERT can be regulated at both the RNA and protein levels. One such regulatory mechanism is TERT turnover. Ubiquitination marks old, damaged, or too concentrated proteins for degradation in the proteasome. In many eukaryotes such as Tetrahymena, Saccharomyces, and humans, TERT is ubiquitinated and processed through the proteasome. Previously, evidence of TERT ubiquitination in Trypanosoma brucei, the parasitic protozoan that causes African sleeping sickness had not been demonstrated. We hypothesized that like other eukaryotes, TbTERT was ubiquitinated, that the absence of TbTR could influence levels of ubiquitination, and that TbTERT was degraded through the proteasome. TbTERT ubiquitination was identified through anti-TbTERT immunoprecipitation followed by an anti-ubiquitin western blot in bloodstream form (BF) and procyclic form (PF) T. brucei. We performed a western blot to determine the polyubiquitin linkage and uncovered K48 polyubiquitination in BF parasites which supports our hypothesis of proteasomal TbTERT degradation. Finally, we administered MG-132 to BF and PF T. brucei to inhibit proteasomal degradation and measured FLAG-tagged TbTERT build-up via western blot. Preliminary data suggests BF and PF T. brucei are ubiquitinated and degraded in the proteasome. Our results indicate that TbTERT ubiquitination likely plays a role in regulating T. brucei telomerase activity which could have implications in cell proliferation rate and disease progression.
References:
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Keywords: Telomerase , Ubiquitination, Regulation