以蛋白質體晶片研究蛋白質亞硫酸化作用

Abstract

酪氨酸硫酸化轉譯後修飾廣泛的作用於許多分泌型蛋白，細胞表面受體和溶酶體酶 中。該修飾藉由調節蛋白質 - 蛋白質相互作用和酶活性誘導細胞內反應。因此，酪氨 酸的硫酸化影響了許多生理和病理過程。生物體中總蛋白質中預計約 1%的酪氨酸殘基 可被硫酸化。然而，目前由實驗證實為蛋白質硫酸化受體非常的少，並且其生物功能 及特徵很多都是未知。在本研究中，我們使用大腸桿菌蛋白酶體微陣列及蛋白質硫酸 化系統來搜尋更多潛在的硫酸化蛋白質受體。硫酸化系統將 PAPS 與 TPST 偶聯以催化 蛋白質受體的酪氨酸硫酸化。本研究中，我們使用大腸桿菌蛋白酶體微陣列作為新的 高通量蛋白質體學方法來篩選和鑑定轉譯後修飾酪氨酸硫酸化的潛在受質。在有 4256 個大腸桿菌 K12 蛋白質的蛋白質體微陣列進行果蠅 TPST 催化的酪氨酸硫酸化修飾。 使用 Cy3 螢光標記的抗體對抗酪氨酸硫酸化抗體的蛋白進行可視化和定量。分析鑑定 的 875 種大腸桿菌硫酸化蛋白質的序列顯示，硫酸化酪氨酸前後 5 個氨基酸的天冬氨 酸和谷氨酸的存在是一個普遍的特徵。該訊息了解酸性氨基酸在轉譯後修飾酪氨酸硫 酸化中扮演重要之角色。轉譯後修飾酪氨酸硫酸化在發炎反應，免疫訊息傳遞激活 化，過氧化自由基反應，病毒感染和可能的許多其他疾病中有著重要作用。可以應用 類似的方法來鑑定酵母和人類蛋白質組的硫酸化蛋白質。我們預計這些方法可能對重 要人類疾病的理解有重大貢獻。為了瞭解亞硫酸化在人體中的重要性，我們針對與細胞生長及凋亡中扮演重要角色的人類表皮生長因子受體與亞硫酸化的關係做進一步的研究及分析。表皮生長因子受體為一高度醣化且具有酪胺酸磷酸酶活性的膜蛋白，表皮生長因子受體激活引發下游的訊息傳遞調控細胞週期，影響細胞生長、分裂和存 活。人類的癌細胞常存在過量的表皮生長因子受體，當其與特定受體結合後可引發眾 多訊息傳導，導致血管新生、細胞轉移及細胞增生並與，導致癌症包括非小細胞肺癌 的形成。透過抑制表皮生長因子受體與特定受體交互作用的抗癌藥物，於治療上有相 當程度的效果，但亦對正常細胞產生影響，導致嚴重的副作用。瞭解表皮生長因子受 體與特定受體之交互作用，將有助於抗癌藥物之發展。我們發現人類表皮生長因子受 體有一新的亞硫酸化轉譯後修飾，經由免疫共沉澱法和西方墨點法，確認表皮生長因 子受體為酪胺酸亞硫酸基轉移酶的受質。由於亞硫酸化轉譯後修飾可以改變蛋白質的 作用，生物體藉由蛋白質亞硫酸化來改變蛋白質性質，藉此調控蛋白質間的交互作用 強度，進而影響到生物之生理功能。

Protein tyrosine sulfation (PTS) is a widespread post-translational modification found in many secreted proteins, a variety of cellular surface receptors and lysosomal enzymes. This modification induces intracellular responses through the regulation of protein-protein interactions and enzymes activities. Therefore, sulfation of tyrosine influences numerous physiological and pathological processes. There are approximately 1% of all tyrosine residues of the total proteins in an organism can be sulfated. However, only a limited amount of sulfated proteins are experimentally demonstrated and their intrinsic features are elusive and remained to be delineated. In this study, we positioned the potential sulfation sites using a protein sulfation system working on E. coli proteasome microarray. The sulfation system coupled in situ PAPS generation with tyrosylprotein sulfotransferase (TPST) to catalyze PTS for potential protein substrates. Herein, we use of a whole E. coli proteasome microarray as a novel high-throughput proteomic approach to screen and identify potential substrates for PTS. Each protein array, which contains 4256 E. coli K12 proteins, was subjected to Drosophila TPST catalyzed tyrosine sulfation modification. Proteins that could be recognized by anti- tyrosine sulfated antibodies were visualized and quantified using Cy3-labeled goat anti-mouse antibodies. Analysis of the sequences of the 875 E. coli sulfated proteins identified revealed that presence of aspartic acid and glutamic acid in less than five amino acid away from the sulfated tyrosine was a general feature. This information constituted a significant addition to the potential proteins subjected to PTS. PTS plays an important role in inflammation reaction, immune signal pathway activation, oxygen stress, virus infection and possible many other diseases. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome chip. We expect such approaches may contribute significantly to the understanding of important human diseases. To understand the importance of PTS in the human, we further researched and analyzed the relation between PTS and Epidermal growth factor receptor (EGFR), which has an important role in cell growth and cell death. EGFR is a membrane protein with a tyrosine kinase activity and a substantial amount of N-liked oligosaccharide. Activation of EGFR triggers anti-apoptotic signaling, proliferation, angiogenesis, invasion, metastasis, and drug resistance, which leads to development and progression of human epithelial cancers, including non-small cell lung cancer. EGFR is often overexpressed in the cancer cells. Numerous signals conduction, resulting in cell metastasis, angiogenesis and other physiological functions are induced following ligand. EGFR is a target of anti-cancer drugs which still have an impact on normal cells, leading to serious side effects. To understand the interaction between ligands and EGFR contribute significantly to the development of anti-cancer drugs. We recently discovered that EGFR is modified through PTS. We also confirmed by Western blotting and immunoprecipitation that EGFR is the substrate of TPST. PTS alter the properties of a protein and significantly affect protein-protein interactions that in turn modify physiological condition in a organism.