In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein

14-3-3σ protein is the most diverged isoform among the seven isoforms of the 14-3-3 protein family. Unlike other 14-3-3 isoforms, its downregulation was demonstrated in various cancer and tumour development, which is in line with its function as an adaptor protein controlling cell cycle and developm...

Full description

Saved in:

Bibliographic Details
Main Author:	Chiang, De Chen
Format:	Thesis
Language:	English
Published:	2023
Subjects:	QD1-999 Chemistry
Online Access:	http://eprints.usm.my/60338/1/CHIANG%20DE%20CHEN%20-%20TESIS24.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-usm-ep.60338
record_format	uketd_dc
spelling	my-usm-ep.603382024-04-03T01:35:40Z In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein 2023-09 Chiang, De Chen QD1-999 Chemistry 14-3-3σ protein is the most diverged isoform among the seven isoforms of the 14-3-3 protein family. Unlike other 14-3-3 isoforms, its downregulation was demonstrated in various cancer and tumour development, which is in line with its function as an adaptor protein controlling cell cycle and development. TRIM25 is an E3 ubiquitin ligase that largely responsible for ubiquitin-mediated protein degradation. Interestingly, 14-3-3σ-TRIM25 interaction leads to 14-3-3σ ubiquitination and subsequent proteasomal degradation. Their exact interacting peptide sequence, however, has yet to be identified. The present study aimed to identify the peptide binding sequence of TRIM25 protein towards 14-3-3σ protein. A total of 5 peptides were identified from in silico bioinformatic analysis (motif search and multiple sequence alignment) for subsequent binding assay. The peptides were either purchased commercially, or synthesised in-house using Fmoc SPPS method, followed by purification using semi-preparative LC and mass-validated with LC-MS. 14-3-3σ protein was successfully expressed using E. coli expression system and purified with affinity chromatography. Interactions between the 6 peptides identified previously and the 14-3-3σ protein expressed were tested with 1H CPMG NMR assay, of which Peptide 1 with sequence 402KLPp(T)FG407 demonstrates the highest binding affinity to 14-3-3σ protein. Competitive CPMG NMR assay of Peptide 1 with Peptide 6 (a known 14-3-3σ binder) revealed substantial binding competition, suggesting that Peptide 1 shares similar binding site with Peptide 6 at the amphipathic pocket 2023-09 Thesis http://eprints.usm.my/60338/ http://eprints.usm.my/60338/1/CHIANG%20DE%20CHEN%20-%20TESIS24.pdf application/pdf en public masters Universiti Sains Malaysia Pusat Pengajian Sains Kimia
institution	Universiti Sains Malaysia
collection	USM Institutional Repository
language	English
topic	QD1-999 Chemistry
spellingShingle	QD1-999 Chemistry Chiang, De Chen In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
description	14-3-3σ protein is the most diverged isoform among the seven isoforms of the 14-3-3 protein family. Unlike other 14-3-3 isoforms, its downregulation was demonstrated in various cancer and tumour development, which is in line with its function as an adaptor protein controlling cell cycle and development. TRIM25 is an E3 ubiquitin ligase that largely responsible for ubiquitin-mediated protein degradation. Interestingly, 14-3-3σ-TRIM25 interaction leads to 14-3-3σ ubiquitination and subsequent proteasomal degradation. Their exact interacting peptide sequence, however, has yet to be identified. The present study aimed to identify the peptide binding sequence of TRIM25 protein towards 14-3-3σ protein. A total of 5 peptides were identified from in silico bioinformatic analysis (motif search and multiple sequence alignment) for subsequent binding assay. The peptides were either purchased commercially, or synthesised in-house using Fmoc SPPS method, followed by purification using semi-preparative LC and mass-validated with LC-MS. 14-3-3σ protein was successfully expressed using E. coli expression system and purified with affinity chromatography. Interactions between the 6 peptides identified previously and the 14-3-3σ protein expressed were tested with 1H CPMG NMR assay, of which Peptide 1 with sequence 402KLPp(T)FG407 demonstrates the highest binding affinity to 14-3-3σ protein. Competitive CPMG NMR assay of Peptide 1 with Peptide 6 (a known 14-3-3σ binder) revealed substantial binding competition, suggesting that Peptide 1 shares similar binding site with Peptide 6 at the amphipathic pocket
format	Thesis
qualification_level	Master's degree
author	Chiang, De Chen
author_facet	Chiang, De Chen
author_sort	Chiang, De Chen
title	In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
title_short	In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
title_full	In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
title_fullStr	In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
title_full_unstemmed	In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein
title_sort	in silico and in vitro characterisation of peptide binding sequence of trim25 cc domain towards 14-3-3 sigma protein
granting_institution	Universiti Sains Malaysia
granting_department	Pusat Pengajian Sains Kimia
publishDate	2023
url	http://eprints.usm.my/60338/1/CHIANG%20DE%20CHEN%20-%20TESIS24.pdf
_version_	1804888916192395264

In Silico And In Vitro Characterisation Of Peptide Binding Sequence Of Trim25 Cc Domain Towards 14-3-3 Sigma Protein

Similar Items