Project Director

Ferdoush, Jannatul

Department Examiner

Beasley, Deanna; Giles, David

Department

Dept. of Biological and Environmental Sciences

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.

Acknowledgments

Primarily, I would like to extend my gratitude to Dr. Deanna Beasley and Dr. David Giles for serving on my departmental honors thesis committee, and for providing their invaluable feedback. Secondly, I would also like to express my gratitude to the following Professors for kindly providing our laboratory with their resources to support this research: Dr. Jose Barbosa (University of Tennessee at Chattanooga), who provided the pRS426 plasmid and XLIB bacterial strain of E. coli, Dr. Sukesh Bhaumik (Southern Illinois University at Carbondale), who provided the pRS406 plasmid, Dr. Daniel Finley (Harvard University), who provided the pUB221 plasmid, as well as Dr. Benjamin Stein (University of Tennessee at Chattanooga), who allowed us to utilize his ChemiDoc machine to visualize our western blots. Our work would be incomplete without all of your contributions. Thirdly, I would like to thank Rizwaan Kadir (lab member of Dr. Ferdoush Lab) for his collaboration in the bioinformatic analyses of Taf2 upregulation in cancer cells. Lastly, I would like to express my sincere gratitude and appreciation to my departmental honors thesis director, Dr. Jannatul Ferdoush, for providing me with this incredible opportunity to learn from and work beside her. I am incredibly thankful for her mentorship, and knowledge that she passionately passes on to her students.

Degree

B. S.; An honors thesis submitted to the faculty of the University of Tennessee at Chattanooga in partial fulfillment of the requirements of the degree of Bachelor of Science.

Date

5-2024

Subject

Cancer cells--Molecular genetics; Eukaryotic cells--Genetics; Genetic transcription--Regulation; Overexpression (Genetics)

Keyword

Taf2; Ubiquitin-Proteasome System; Overexpression

Discipline

Molecular Genetics

Document Type

Theses

Extent

ii, 46 leaves

DCMI Type

Text

Language

English

Rights

http://rightsstatements.org/vocab/InC/1.0/

License

http://creativecommons.org/licenses/by-sa/4.0/

Date Available

5-1-2024

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