利用化學探針分析蛋白質酪胺酸磷酸水解酶

蛋白質酪胺酸磷酸為細胞裡一種常見的蛋白質轉譯後修飾，此修飾為一種動態平衡的化學反應，主要是由蛋白質酪胺酸激酶以及蛋白質酪胺酸磷酸水解酶所調控。蛋白質酪胺酸磷酸水解酶為細胞中一種重要的酵素族群，主要是催化蛋白質中磷酸化酪胺酸其磷酸根的水解，調控許多生理反應，例如細胞的複製或是分化等。傳統上研究細胞裡酪胺酸磷酸水解酶的含量以及變化可以藉由質譜儀的幫助進行分析，但是此方式無法精準地反映出酵素的活性變化為何，因此較不利於研究的發展。為了解決這個問題，以活性為基礎的化學探針可做為一有效的分析工具，提供研究蛋白質酪胺酸磷酸水解酶其活性的分析平台。針對酪胺酸磷酸水解酶此酵素族群，我們以馬來醯亞胺、馬來酸以及延胡索酸做為化學探針的辨識端，去探討這些化學探針對於酪胺酸磷酸水解酶的標定反應。首先，我們以一種酪胺酸磷酸水解酶做為模式並進行標定實驗探討，我們發現以馬來酸為辨識端的化學探針對於酪胺酸磷酸水解酶的標定能力較強，因此以此結構進行後續的實驗，我們發現此結構可進入酵素的活化位與其催化性胺基酸形成共價鍵結，而此結構也可標定到不同類型的酪胺酸磷酸水解酶；在專一性的部分，以馬來酸為辨識端的化學探針除了會標定酪胺酸磷酸水解酶以外，還是會標定到其他非酪胺酸磷酸水解酶的酵素，因此該結構仍具有非專一性標定的問題存在。此外，實驗室先前已經成功發展出以mchanism-based為基礎並可專一地標定酪胺酸磷酸水解酶的活性化學探針，而本篇的另一個研究部分主要是以此化學探針做為一分析工具去分析細胞裡雙專一性磷酸水解酶的活性，以此建立一個能分析雙專一性磷酸水解酶活性的平台。在本研究中，我們以DUSP14做為一個模式，於不同的生物體系統下過度表現此酵素，使用活性化學探針並且搭配免疫沉澱分析法，我們成功地將DUSP14從複雜的蛋白質體中分離出來，同時也可以直接地偵測並分析此酵素的活性狀態。

關鍵字

蛋白質酪胺酸磷酸水解酶；活性化學探針；馬來醯亞胺；馬來酸；延胡索酸；雙專一性磷酸水解酶

並列摘要

Protein tyrosine phosphorylation is a common post-translational modification of proteins. It is a dynamic, reversible process which is regulated by protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPs). PTP is an important enzyme family that regulates tyrosine phosphorylation status in cells. Although the protein levels of PTPs can be determined by mass spectrometry, the analysis cannot precisely profile the functional state of PTPs in living cells. To overcome the hurdle, activity-based protein profiling (ABPP) was introduced as a tool to profile PTP activities. Here, we have synthesized a series of maleimide-related probes using maleimide, maleate, and, fumarate as the recognition moiety. We evaluate the labeling efficiency and specificity of these probes using purified PTPs. We found the maleate-based chemical probes reacted with the catalytic residue of tyrosine phosphatse and labeled different tyrosine phosphatases. However, since these probes also labeled other non-PTPs, the specificities of these probes have to be further improved. In addition, we have also used a 2-fluoromethyl phosphotyrosine (2-FMPT), which was developed previously in our lab, to establish an activity measuring system for dual-specific phosphatase (DUSP). Using DUSP14 as the model, we found the activity of DUSP14 can be detected directly in cell lysates through combination of activity probe-labeling and immunoprecipitation using a specific antibody against DUSP14.

並列關鍵字

protein tyrosine phosphatase ； activity-based chemical probe ； maleimide ； maleate ； fumarate ； dual-specific phosphatase

參考文獻

Hunter, T., Dixon, J., and Mustelin, T. (2004). Protein Tyrosine Phosphatases in the Human Genome.

Arena, S., Benvenuti, S., and Bardelli, A. (2005). Genetic analysis of the kinome and phosphatome

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D. M. (2006). TAK1-binding protein 1 is a pseudophosphatase. Biochem J, 399, 427-434.

Denu, J. M., and Dixon, J. E. (1998). Protein tyrosine phosphatases: mechanisms of catalysis and

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利用化學探針分析蛋白質酪胺酸磷酸水解酶

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