Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity
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Kent State University Honors College / OhioLINK
2020
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-ksuhonors15882722509500922021-08-03T07:14:50Z Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity Schmocker, Stefani P. Biochemistry FRET FTO TLC N6-methyladenosine demethylase The methylation of the sixth position of adenine (m<sup>6</sup>A) is an epitranscriptomic modification that has been found to play a vital role in early cell differentiation and embryonic development. The removal of the m<sup>6</sup>A modification is facilitated by the "eraser" protein FTO. The protein FTO (fat mass and obesity-associated protein) is a non-heme Fe<sup>2+</sup> and α-ketoglutarate-dependent dioxygenase demethylase enzyme whose activity has been recently linked to spermatogenesis and cancer progression. However, little is known about the biophysical nature of its binding to its RNA targets. First, FTO was produced in BL21 (DE3) Escherichia coli transformed with the PET45-C<sup>+1</sup> plasmid and purified using affinity chromatography. Thin Layer Chromatography with<sup>32</sup> P-labeled, m<sup>6</sup>A-containing target RNA which was incubated with FTO resulted in loss of the m<sup>6</sup>A modification, as indicated by changes in the spotting pattern compared to a control reaction. Additionally, FRET experiments were used to determine the effect of FTO's cofactors (α-ketoglutarate and Fe<sup>2+</sup>) on its binding thermodynamics, revealing slightly increased dissociation constant (K<sub>D</sub>) values in the absence of each cofactor (α-ketoglutarate and Ni<sup>2+</sup>, an Fe<sup>2+</sup> analog). This suggests that FTO binding to m<sup>6</sup>A RNA is slightly strengthened when both cofactors are present in solution. FRET experiments also indicated the potential of a phage-display isolated peptide to serve as an inhibitor of FTO's demethylase activity. 2020-05-26 English text Kent State University Honors College / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092 http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092 restricted--full text not available online This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
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English |
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NDLTD |
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Biochemistry FRET FTO TLC N6-methyladenosine demethylase |
| spellingShingle |
Biochemistry FRET FTO TLC N6-methyladenosine demethylase Schmocker, Stefani P. Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| author |
Schmocker, Stefani P. |
| author_facet |
Schmocker, Stefani P. |
| author_sort |
Schmocker, Stefani P. |
| title |
Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| title_short |
Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| title_full |
Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| title_fullStr |
Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| title_full_unstemmed |
Investigation of the Binding of FTO to the N6-methyladenosine Modification and Evaluating the Ability of the MTR-pep1 Peptide to Inhibit its Demethylase Activity |
| title_sort |
investigation of the binding of fto to the n6-methyladenosine modification and evaluating the ability of the mtr-pep1 peptide to inhibit its demethylase activity |
| publisher |
Kent State University Honors College / OhioLINK |
| publishDate |
2020 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1588272250950092 |
| work_keys_str_mv |
AT schmockerstefanip investigationofthebindingofftotothen6methyladenosinemodificationandevaluatingtheabilityofthemtrpep1peptidetoinhibititsdemethylaseactivity |
| _version_ |
1719457130482761728 |