Site-Specific and Enzyme-Specific Acetylproteome

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簡歷

基本資料

Project title

Code/計畫編號

MOST106-2113-M019-001

Translated Name/計畫中文名

具位點特異性與酵素特異性之乙醯化蛋白體

Project Coordinator/計畫主持人

Pang-Hung Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=12243068

Year

2017

Start date/計畫起

01-08-2017

Expected Completion/計畫迄

31-07-2018

Bugetid/研究經費

1100千元

ResearchField/研究領域

化學

在生物體中乙醯化修飾是非常重要的一種蛋白質後轉譯修飾，乙醯基可藉由乙醯轉移酶的作用修飾至賴氨酸的側鏈氨基上，而去乙醯酶則是作為細胞內蛋白質乙醯化修飾的負調控。乙醯化修飾對於細胞內的基因表達與代謝調控上具有非常重要的影響，而失去調控的蛋白質乙醯化往往對細胞造成嚴重影響，甚至導致細胞癌化。目前乙醯化蛋白體學的研究，仍採用以泛乙醯化抗體富集細胞中具乙醯化修飾之蛋白質或胜肽，再配合液相層析串聯式質譜法進行分析的方法為主。而採用此免疫沈澱法的缺點在於無法確知泛乙醯化抗體的富集效率，且無法得知此乙醯化的位點是經由何種乙醯轉移酶或去乙醯酶所調控。為改良傳統乙醯化蛋白體學的研究方法，本計畫提出一嶄新的蛋白體分析策略，以細胞外酵素反應配合化學標記，達到可同時鑑定蛋白質中乙醯化位點並確認調控此修飾的酵素。本計畫預計以三年時間完成以下主要目標：(1) 開發分析具去乙醯酶專一性之乙醯化蛋白體學方法；(2) 開發由特定乙醯轉移酶所調控之乙醯化蛋白體學方法；(3) 建立具位點特異性與酵素特異性之乙醯化蛋白體網絡。本計畫的成果對於瞭解細胞內乙醯化調控的機制將產生全面性的影響，特別是在確知特定去乙醯酶調控的目標蛋白中乙醯化的位點，即可從分子層級明瞭去乙醯酶抑制劑如何影響腫瘤細胞生長，對於癌症治療的相關研究上將極有助益。Protein acetylation is one of the important post-translational modifications (PTMs) in organisms. The acetyl group is transferred from acetyl coenzyme A (Ac-CoA) to the amino group of lysine side chain by lysine acetyltransferase. Deacetylase, on the other hand, is responsible for the removal of acetyl group in cells. Acetylation is tightly correlated with gene regulation and cellular metabolism; moreover, the dysregulational acetylation leads to serious consequence, such as oncogenesis. The current acetylproteomics study is based on the enrichment of acetylated proteins or peptides by pan-anti-acetyllysine antibody followed by the LC-MS analysis. However, two major downsides of this popular method are mentioned, including how general the pan-antibody is for interacting with all different kinds of acetylated targets, and which acetyltransferase and deacetylase are involved in regulation. In order to overcome these two drawbacks, we propose a novel proteomics method based on the in vitro enzyme assay coupled with chemical labeling followed by LC-MS analysis. Not only the acetylated sites can be correctly identified, but also the upstream acetyltransferase and downstream deacetylase for regulation can be determined. Three major aims for this proposal are: (1) to develop an in vitro deacetylase assay for deacetylase-specific acetylproteomics; (2) to develop an in vitro acetyltransferase assay for enzyme-specific acetylproteomics; (3) to construct a site-specific and enzyme-specific comprehensive acetylproteomics interaction map. We believe that this new approach can help us to understand detailed mechanism of acetylation from molecular level and provide a new vision in acetylproteomics studies.

Keyword(s)

Protein Acetylation
Deacetylase
Acetyltransferase
Acetylproteomics
乙醯化修飾
去乙醯酶
乙醯轉移酶
乙醯化蛋白體學

DSpace CRIS

Site-Specific and Enzyme-Specific Acetylproteome

基本資料

Description