利用內生蛋白質降解系統進行高通量蛋白羧基端可及性試驗

Abstract

蛋白質羧基端與胺基端對於其功能的調控是重要的修飾位點。蛋白質端點修飾常常藉變其在細胞中其他分子之於他的可及性。因此研究蛋白質端點的可及性不僅可以用來探討蛋白質特性，也能偵測蛋白質活動。膜蛋白的相關研究特別仰賴蛋白質端點可及性的試驗，因為膜蛋白的方向，即膜蛋白拓撲，與他的功能是相輔相成的。常見的膜蛋白方向試驗技術是勞力高度技術性。近來學者發現了隸屬泛素-蛋白酶體系統的蛋白質羧基端依賴降解途徑。E3 泛素連接酶複合體中的受體負責受質的專一性選擇，並且標定帶有特定降解訊號 的蛋白質。因此我利用這個降解機制建立了一套技術，用以研究蛋白質羧基端的可及性。接上羧基端降解訊號的蛋白質會被標定並且位在細胞質或是細胞核中才會被降解。這個試驗可以被應用在分析膜蛋白的羧基端拓撲基於分佈且被隔離在不同膜狀胞器與利用可及性的差異探討細胞質蛋白質的結構。實驗數據顯示這個試驗不僅提供可靠的判讀結果，也允許彈性的、蛋白質相關的技術組合。我更進一步用這套技術執行高通量膜蛋白拓撲的分析。這個基於內生性系統開發的技術易於操作、在不犧牲準確度、靈敏度的前提下成功提高膜蛋白拓撲分析的試驗量。

Protein termini are important sites for functions. The regulations of termini are often related to changes of their accessibility to other molecules in cells. Therefore, examining accessibility of protein terminus can be not only an indicator for protein features but also a sensor for activities. Studies of transmembrane proteins are highly requested of terminus analysis because their orientation, membrane protein topology, is largely coupled with their functions. Common membrane protein topology assay like protease protection assay is laborious and depends heavily on skillful manipulation. Recently, a C-terminal end-directed protein degradation pathway in ubiquitin-proteasome system was found. The receptor in E3 ubiquitin ligase complex is responsible for substrate selection and it targets protein C-termini featuring specific degradation signal. I developed an assay relying on this pathway to analyze accessibility of protein C-terminus. Proteins fused with a C-end degradation signal would be targeted and degraded only if their C-terminus is both localized and exposed in cytosol. This assay can be applied to examine C-terminal topology for membrane proteins based on separated subcellular localization and also C-terminal conformation depending on its accessibility for cytosolic proteins. Proof of principle experiments suggested this assay is reliable and flexible for alternative readouts, and I further conducted high throughput membrane protein topology analysis. This physiological system-based technique is simple in manipulation, allows higher throughput without sacrificing accuracy and sensitivity, and potentially can be applied in studying protein dynamic regulation in living cells.