Publisher
University of Tennessee at Chattanooga
Place of Publication
Chattanooga (Tenn.)
Abstract
Through several studies, Taf2 has been found to be upregulated in various cancer cells. However, the mechanism through which this increased expression of Taf2 occurs remains unknown. As evolutionarily conserved ubiquitin-proteasome system (UPS) maintains protein homeostasis for normal cellular function, we hypothesized that stability of Taf2 may be regulated by this UPS and this UPS may be dysregulated in cancer cells causing overexpression of Taf2. To test our hypothesis, we assessed the role of the UPS in the regulation of the stability of Taf2 by 26S proteasome-mediated degradation. To do so, we performed molecular experiments mainly through two steps: 1st step includes the analysis of the ubiquitination status of Taf2, and 2nd step includes the analysis to determine if Taf2 is regulated by the 26S proteasome. Similarly to UPS, Taf2 is evolutionarily conserved and found in both yeast and humans as we conducted these experiments in yeast (Saccharomyces cerevisiae). To perform the 1st step, we conducted molecular cloning to introduce the pUB221 plasmid, expressing the hexahistidine-tagged ubiquitin under the CUP1 promoter, into the yeast strain containing TAP-tagged Taf2. Next, using this strain, we performed Ni2+- NTA-based ubiquitination assay to see if Taf2 is regulated by ubiquitination or not. Notably, for the 1st time we found that Taf2 undergoes polyubiquitination. Generally, if a protein undergoes polyubiquitination, it is likely to be degraded by the 26S proteasome. To evaluate this possibility, we investigated the activity of the 26S proteasome in regulating Taf2 stability. Briefly, we performed MG132 based proteasomal inhibition assay (2nd step). MG132 inhibits the proteolytic function of the 26S proteasome and therefore, if Taf2 is regulated by the proteasome we would observe increased abundance of Taf2 following proteasomal inhibition by MG132. However, yeast cells contain the multidrug resistance gene, therefore, to perform the MG132 based proteasomal inhibition assay, we knocked out of the multidrug resistance gene, PDR5, in the yeast strain expressing TAP-tagged Taf2, and treat these cells with MG132. Importantly, we found that polyubiquitylated Taf2 is targeted for degradation by the 26S proteasome. Our results showed for the first time that Taf2 abundance is regulated by the UPS. In addition, our bioinformatics data suggest that protein overexpression, as opposed to mRNA overexpression, plays a role in several cancers implicating a mismanaged UPS system. Thus, our results reveal novel UPS regulation of Taf2 with potentials for future therapeutic intervention.
Document Type
posters
Language
English
Rights
http://rightsstatements.org/vocab/InC/1.0/
License
http://creativecommons.org/licenses/by/4.0/
Recommended Citation
Osborn, Morgan, "Elucidation of the Overexpression of Taf2 in Eukaryotic Cells". ReSEARCH Dialogues Conference proceedings. https://scholar.utc.edu/research-dialogues/2024/Proceedings/8.
Elucidation of the Overexpression of Taf2 in Eukaryotic Cells
Through several studies, Taf2 has been found to be upregulated in various cancer cells. However, the mechanism through which this increased expression of Taf2 occurs remains unknown. As evolutionarily conserved ubiquitin-proteasome system (UPS) maintains protein homeostasis for normal cellular function, we hypothesized that stability of Taf2 may be regulated by this UPS and this UPS may be dysregulated in cancer cells causing overexpression of Taf2. To test our hypothesis, we assessed the role of the UPS in the regulation of the stability of Taf2 by 26S proteasome-mediated degradation. To do so, we performed molecular experiments mainly through two steps: 1st step includes the analysis of the ubiquitination status of Taf2, and 2nd step includes the analysis to determine if Taf2 is regulated by the 26S proteasome. Similarly to UPS, Taf2 is evolutionarily conserved and found in both yeast and humans as we conducted these experiments in yeast (Saccharomyces cerevisiae). To perform the 1st step, we conducted molecular cloning to introduce the pUB221 plasmid, expressing the hexahistidine-tagged ubiquitin under the CUP1 promoter, into the yeast strain containing TAP-tagged Taf2. Next, using this strain, we performed Ni2+- NTA-based ubiquitination assay to see if Taf2 is regulated by ubiquitination or not. Notably, for the 1st time we found that Taf2 undergoes polyubiquitination. Generally, if a protein undergoes polyubiquitination, it is likely to be degraded by the 26S proteasome. To evaluate this possibility, we investigated the activity of the 26S proteasome in regulating Taf2 stability. Briefly, we performed MG132 based proteasomal inhibition assay (2nd step). MG132 inhibits the proteolytic function of the 26S proteasome and therefore, if Taf2 is regulated by the proteasome we would observe increased abundance of Taf2 following proteasomal inhibition by MG132. However, yeast cells contain the multidrug resistance gene, therefore, to perform the MG132 based proteasomal inhibition assay, we knocked out of the multidrug resistance gene, PDR5, in the yeast strain expressing TAP-tagged Taf2, and treat these cells with MG132. Importantly, we found that polyubiquitylated Taf2 is targeted for degradation by the 26S proteasome. Our results showed for the first time that Taf2 abundance is regulated by the UPS. In addition, our bioinformatics data suggest that protein overexpression, as opposed to mRNA overexpression, plays a role in several cancers implicating a mismanaged UPS system. Thus, our results reveal novel UPS regulation of Taf2 with potentials for future therapeutic intervention.