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postgraduate thesis: Developing chemical tools to study fatty-acylation and histone crotonylation

TitleDeveloping chemical tools to study fatty-acylation and histone crotonylation
Authors
Advisors
Advisor(s):Li, XD
Issue Date2017
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Li, X. [李歆]. (2017). Developing chemical tools to study fatty-acylation and histone crotonylation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractProtein posttranslational modification (PTM), normally referring to the covalent attachments of small chemical tags onto proteins after their biosynthesis, is one of the cellular mechanisms adopted by the cells to expand the functional diversity of the proteome. The protein PTMs are involved in the regulation of essentially every cellular process. To date, more than two hundred of protein PTMs have been identified. Except for the several well-studied ones, for example acetylation, phosphorylation, and methylation, we have limited knowledge for most of them on their regulatory mechanisms and biological significances. My research during the past four years mainly focused on the development of chemical tools to study the protein fatty-acylation and histone lysine crotonylation (Kcr). Protein fatty-acylation plays important roles in the regulation of trafficking and membrane targeting of the substrate proteins. In the last decade, the development of chemical reporters for fatty-acylation have facilitated the identification of fatty-acylated proteins. However, the systematic mapping of fatty-acylated sites in substrate proteins remains challenging, largely due to the poor behaviors caused by the high hydrophobicity of the fatty-acylated proteins/peptides in LC-MS analysis. To overcome this difficulty, in Chapter 2, I developed cleavable selenoether-functionalized chemical reporters for the mapping of protein fatty-acylated sites. The selenoether linkage is readily cleaved under mild oxidative condition, cutting off the long fatty acyl chain. The hydrophobicity of the proteins/peptides is therefore decreased to facilitate the LC-MS analysis. However, the study was ended up without achieving the initial goal, as the reporters were either with poor performances in the bioorthogonal reactions, or failed to be incorporated into the proteins. Previous study of our group identified that Sirt3, an NAD-dependent lysine deacetylase, was a specific binding partner of a crotonylation mark at histone H3 Lys4 (H3K4cr). In Chapter 3, Sirt3 was showed to catalyze the hydrolysis of crotonyllysine using a similar mechanism as its deacetylation. In the previously resolved crystal structure of Sirt3-H3K4cr complex, the crotonyl group forms a pi-pi interaction with the Phe180 residue of Sirt3. In this study, mutagenesis studies were carried out to examine the roles played by this pi-pi interaction in the recognition and hydrolysis of Kcr. The decrotonylase activity of other sirtuins were also tested. The results showed that Sirt1 and Sirt2 also pose robust decrotonylase activity in vitro. In Chapter 4, a collection of peptide-based inhibitor of AF9 YEATS domain were designed and synthesized. The YEATS domain is a newly identified reader of histone Kcr, recognizing the crotonyl group through a pi-pi-pi stacking in the binding pocket. The inhibitors were designed to target this pi-pi-pi stacking by replacing the crotonyl group with different pi system-containing groups. Several potent inhibitors with nanomolar level dissociation constants were acquired. Molecular docking study were performed to investigate the AF9-inhibitoe interaction. The selectivity of inhibitors among other YEATS domains were also examined.
DegreeDoctor of Philosophy
SubjectAcylation
Lysine
Sirtuins
Dept/ProgramChemistry
Persistent Identifierhttp://hdl.handle.net/10722/254037

 

DC FieldValueLanguage
dc.contributor.advisorLi, XD-
dc.contributor.authorLi, Xin-
dc.contributor.author李歆-
dc.date.accessioned2018-06-05T05:52:59Z-
dc.date.available2018-06-05T05:52:59Z-
dc.date.issued2017-
dc.identifier.citationLi, X. [李歆]. (2017). Developing chemical tools to study fatty-acylation and histone crotonylation. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/254037-
dc.description.abstractProtein posttranslational modification (PTM), normally referring to the covalent attachments of small chemical tags onto proteins after their biosynthesis, is one of the cellular mechanisms adopted by the cells to expand the functional diversity of the proteome. The protein PTMs are involved in the regulation of essentially every cellular process. To date, more than two hundred of protein PTMs have been identified. Except for the several well-studied ones, for example acetylation, phosphorylation, and methylation, we have limited knowledge for most of them on their regulatory mechanisms and biological significances. My research during the past four years mainly focused on the development of chemical tools to study the protein fatty-acylation and histone lysine crotonylation (Kcr). Protein fatty-acylation plays important roles in the regulation of trafficking and membrane targeting of the substrate proteins. In the last decade, the development of chemical reporters for fatty-acylation have facilitated the identification of fatty-acylated proteins. However, the systematic mapping of fatty-acylated sites in substrate proteins remains challenging, largely due to the poor behaviors caused by the high hydrophobicity of the fatty-acylated proteins/peptides in LC-MS analysis. To overcome this difficulty, in Chapter 2, I developed cleavable selenoether-functionalized chemical reporters for the mapping of protein fatty-acylated sites. The selenoether linkage is readily cleaved under mild oxidative condition, cutting off the long fatty acyl chain. The hydrophobicity of the proteins/peptides is therefore decreased to facilitate the LC-MS analysis. However, the study was ended up without achieving the initial goal, as the reporters were either with poor performances in the bioorthogonal reactions, or failed to be incorporated into the proteins. Previous study of our group identified that Sirt3, an NAD-dependent lysine deacetylase, was a specific binding partner of a crotonylation mark at histone H3 Lys4 (H3K4cr). In Chapter 3, Sirt3 was showed to catalyze the hydrolysis of crotonyllysine using a similar mechanism as its deacetylation. In the previously resolved crystal structure of Sirt3-H3K4cr complex, the crotonyl group forms a pi-pi interaction with the Phe180 residue of Sirt3. In this study, mutagenesis studies were carried out to examine the roles played by this pi-pi interaction in the recognition and hydrolysis of Kcr. The decrotonylase activity of other sirtuins were also tested. The results showed that Sirt1 and Sirt2 also pose robust decrotonylase activity in vitro. In Chapter 4, a collection of peptide-based inhibitor of AF9 YEATS domain were designed and synthesized. The YEATS domain is a newly identified reader of histone Kcr, recognizing the crotonyl group through a pi-pi-pi stacking in the binding pocket. The inhibitors were designed to target this pi-pi-pi stacking by replacing the crotonyl group with different pi system-containing groups. Several potent inhibitors with nanomolar level dissociation constants were acquired. Molecular docking study were performed to investigate the AF9-inhibitoe interaction. The selectivity of inhibitors among other YEATS domains were also examined. -
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshAcylation-
dc.subject.lcshLysine-
dc.subject.lcshSirtuins-
dc.titleDeveloping chemical tools to study fatty-acylation and histone crotonylation-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineChemistry-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2017-
dc.identifier.mmsid991043962781203414-

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