Application of SNAP-TAG technology for observing sumoylation dynamics during keratinocyte differentiation and the alterations caused by human papilloma virus E6 proteins : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2011]
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| Abstract: | ABSTRACT: SNAP-tag technology is a protein labeling technique that is derived from the DNA repair enzyme O⁶-alkylguanine-DNA alkytransferase with its fluorescent substrates being derivatives of benzyl purines and benzyl pyrimidines. Using this technology we developed cell lines expressing SNAP-tagged SUMO3 for the evaluation of the sumoylation system during keratinocyte differentiation. Previously our lab demonstrated that overall sumoylation increases over the course of keratinocyte differentiation, and SUMO3 was chosen for our studies as expression of this modifier increase during keratinocyte differentiation. Our resluts indicate modification of proteins by SUMO3 during keratinocyte differentiation is a highly dynamic process and suggest that sumoylation changes in individual substrate proteins may be important for the differentiation process. SUMO3 was shown to decrease sharply then exhibit a steady rise until the last time point was taken. These results show that not only is SNAP-tag technology a viable option for studying SUMO modification, but that individual isoforms may have varying roles in the differentiation process. Papillomaviruses are double stranded DNA viruses whose life cycle is tied to the differentiation of the keratinocytes and mucosal epithelia they infect. It has been demonstrated that papillomavirus proteins E1, E2, and L2 are targets of the sumoylation system. Perhaps more interesting is the demonstration that high risk E6 effects PIASy protein, a SUMO E3 ligase. Preliminary studies in our lab have shown E6 interacts with and causes a loss of Ubc9 in 293 cells. In this study we applied SNAP-tag technology in conjunction with HPV 16E6 expressing cell lines to determine the ability of the E6 protein to perturb the sumoylation and affect differentiation. Our results demonstrate that E6 protein effectively reduces sumoylation of targets most likely through degradation of Ubc9 and that this may prevent proper differentiation. In conclusion we demonstrate that SNAP-tag technology is a viable option for detecting sumoylated proteins, and point to the E6 protein as having a significant role in altering the sumoylation system. |
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| Item Description: | "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of Texas A&M University System Health Science Center in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2011." Approved as to style and content by: Van G Wilson, Julian Leibowitz, Warren E. Zimmer, Steve A. Maxwell, James E. Samuel Vita. |
| Physical Description: | xiv, 234 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references. |