Date Thesis Awarded

5-2017

Document Type

Honors Thesis

Degree Name

Bachelors of Science (BS)

Department

Biology

Advisor

Lizabeth Allison

Committee Member

Matthew Wawersik

Committee Member

William Buchser

Committee Member

Jennifer Stevens

Abstract

The small ubiquitin-like modifier (SUMO) is involved in post-translational modification of proteins and is characterized by its role in regulation of a variety of cellular processes. The objective of this thesis was to characterize the role SUMO has in modification of the thyroid hormone receptor (TR). TR is a regulatory transcription factor that, in most cases when the ligand (T3) is not bound, represses gene expression through the recruitment of co-repressors. When T3 is bound to TR, the co-repressors are released and co-activators are recruited, resulting in positive gene expression mediated by a particular thyroid hormone response element (TRE). Previous research has identified the import and export proteins involved in nuclear localization of TR. Post-translational modification, however, has only begun to be characterized. In terms of proteolysis, TR is degraded through poly-ubiquitination; but it is unclear whether ubiquitin binding is correlated with sumoylation. SUMO sites for TR have been previously identified, and their role in TR-mediated gene expression demonstrated. Here, the role SUMO plays in nuclear localization and proteolysis of TR through its interaction with ubiquitin binding is characterized. Mutant constructs of TR that could not be sumoylated were synthesized and cloned into GFP. For nuclear localization assays, the constructs were transfected into HeLa cells and quantitatively scored for the ratio of fluorescence intensity in the nucleus versus the cytosol using region of interest (ROI) analysis. Coimmunoprecipitation followed by western analysis, was conducted in order to identify the relationship between sumoylation and ubiquitination. The nuclear localization experiments showed no changes in nuclear localization of the SUMO-deficient TR compared to the wild-type TR. Coimmunoprecipitation suggested that there may be a higher level of ubiquitination when TR was not sumoylated. However, varying levels of cellular ubiquitin make this finding inconclusive. Overall, this thesis demonstrates that SUMO is not directly involved in nuclear localization of TR; however, it may play a role in enhancing the binding of ubiquitin, ultimately suggesting that sumoylation may be involved in proteolysis of TR.

Available for download on Friday, May 10, 2019

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