半胱胺酸磺酸化誘發酪氨酸去磷酸酶以泛素化降解之研究: 氧化壓力下蛋白質品質控管之分子機制

Cysteine sulfonation induces ubiquitin-dependent proteolysis of protein tyrosine phosphatases: A molecular basis for protein quality control under oxidative stress

楊君怡(Chun-Yi Yang)

指導教授 ： 孟子青

國立臺灣大學/生命科學院/生化科學研究所/博士(2018年)

https://doi.org/10.6342/NTU201802872

摘要

活性氧化物質在心臟病生理學的發展和進展中扮演關鍵作用。蛋白質是心肌細胞中產生的細胞活性氧化物質的主要標定之一。在20種常見的胺基酸中，半胱胺酸因為具有低解離常數值特性所以非常容易進行氧化反應。基於半胱胺酸座落在蛋白質功能基位點，而這類型蛋白質於病生理中扮演著重要的角色，所以顯示出定義硫醇修飾的氧化還原作用角色的重要性。在本論文裡，我們的研究重心探討的是不可逆半胱胺酸磺酸化會對於蛋白質酪氨酸磷酸水解酶進行氧化還原的調控。蛋白質酪氨酸磷酸水解酶活化位點的半胱胺酸具有低解離常數值特性,因此這類酵素的半胱胺酸也很容易被內生性活性氧化物質直接標定。為了研究在有活性氧化物質誘導下，半胱胺酸磺酸化對於蛋白質酪氨酸磷酸水解酶的影響以及蛋白質品量控管機制下蛋白水解的機制。我們使用了可以直接偵測蛋白質半胱胺酸磺酸化的特定抗體，並且發現在H9c2細胞中內生性蛋白質酪氨酸磷酸水解酶不可逆氧化的存在並且會走向蛋白酶體的降解。蛋白質酪氨酸磷酸水解酶1B（PTP1B）是這類酵素中在半胱胺酸氧化還原研究方面較完整，所以我們選擇PTP1B作為我們的實驗模型。透過細胞內和細胞外免疫沉澱分析方法，我們發現在有H2O2的處理下,PTP1B活化位點Cys215會被磺酸化，而這類型的氧化修飾進而有利於PTP1B泛素化修飾與其蛋白水解。然而我們將PTP1B活化位點Cys215進行性定點突變則會抑制PTP1B被磺酸化進而消除了PTP1B泛素化修飾與其降解。進一步我們透過高通量酵母菌蛋白質體晶片，我們找出與人類蛋白質Cul1 E3連接酶同源的酵母菌蛋白質CDC53可以和具有半胱胺酸磺酸化的化學合成胜肽進行強烈地交互作用。我們隨後在Cul1 E3連接酶的顯性失活結構體實驗顯示出透過Cul1對於PTP1B活化位點Cys215磺酸化的辨認進而將PTP1B視為Cul1的受質。當我們抑制Cul1針對半胱胺酸磺酸化蛋白質酪氨酸磷酸水解酶的蛋白質降解會則增加心臟毒性的產生。這些結果提供了生物學上的半胱胺酸氧化修飾意義，而這種氧化修飾會促使心肌蛋白質經由泛素/蛋白酶體途徑進行蛋白質代谢。

關鍵字

心臟 ； 半胱胺酸修飾 ； 半胱胺酸磺酸化 ； 蛋白質酪氨酸磷酸水解? ； 蛋白質降解 ； 泛素/蛋白?體機制

並列摘要

Reactive oxygen species (ROS) has been shown to play a critical role in the development and progression of cardiac pathophysiology. Protein is one of the prime targets of cellular ROS generated in cardiomyocytes. Among the 20 common amino acid, cysteine (Cys) residue, which has a remarkably low pKa characteristic, is highly susceptible to oxidation. Identification of the redox role of thiol modifications has gained significant importance because Cys residue plays an important role in protein function under pathophysiological conditions. Here, our study focuses on the redox-dependent regulation of Cys-sulfonation (SO3H), which belongs irreversible oxidation, on protein tyrosine phosphatases (PTPs). Because of its high nucleophilic property, catalytic Cys residue within PTPs is the direct targets of cellular ROS. To investigate the effect of ROS-induced Cys-sulfonation on PTPs and the mechanism of proteolysis in protein quality control machinery. Using the antibody-based method that could detect protein Cys-sulfonation, we found that endogenous PTPs already oxidized irreversibly and undergoing proteasome-mediated degradation in H9c2 cells. Protein tyrosine phosphatase 1B (PTP1B) is one of PTPs oxidation well-studied, so we chose PTP1B as our experiment model. Upon H2O2 stimuli, through the in vivo and in vitro immunoprecipitation assays, we found that PTP1B catalytic Cys215 residue was sulfonated and it facilitated PTP1B for ubiquitination and proteolysis. Inhibition of PTP1B oxidation by site-directed mutagenesis of PTP1B at active-site Cys215 abrogates the effects of protein ubiquitination and degradation on PTP1B. Through the high-throughput Yeast proteome chips, we identified CDC53, which has homology to human Cul1 E3 ligase, could strongly interact with the chemically synthesized peptide containing Cys-sulfonation. Subsequent investigation with the dominant-negative construct of Cul1 E3 ligase suggested that PTP1B serve as the substrate for Cul1 via Cys-sulfonation recognition. Impairment of Cul1-mediated degradation of Cys-sulfonated PTPs potentiated cardiotoxicity. The findings of this study provide important insight into how biologically significant Cys oxidation directs myocardial protein turnover through ubiquitin/proteasome-mediated degradation.

並列關鍵字

Heart ； Cys modification ； Cys-sulfonation ； Protein tyrosine phosphatase ； Protein degradation ； Ubiquitin/proteasome mechanism

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