利用螢光共振能量轉移技術於活細胞內分析蛋白酶之作用機轉

Abstract

蛋白質降解是細胞中正常蛋白質代謝的一個重要過程，蛋白酶不只參與在蛋白質分解的過程中，在蛋白質活化、細胞生長、分裂、分化、移動、訊息傳遞及其他細胞的生理活動中，都扮演重要角色。此外，許多病毒利用細胞或病毒本身的蛋白酶進行活化本身表面蛋白或參與在細胞膜融合的過程。很自然地，蛋白酶抑制劑就成為極具吸引力的抗病毒藥物。雖然在最近幾年已經利用各種不同蛋白酶特性在細胞外進行許多活性的研究，但是要在活細胞中直接研究其活性仍有許多困難待克服。 在本篇論文中，我們嘗試建立一個利用螢光共振能量轉移的技術，在活細胞中研究蛋白酶活性的平台。首先構築一個黃色螢光蛋白-藍色螢光蛋白的融合蛋白，並在兩蛋白質間留下多位轉殖區以供後續構築特殊蛋白酶切割辨識序列之用。當一個特殊蛋白酶切割辨識序列正確地構築在多位轉殖區中，一個特殊的蛋白酶受質蛋白質就完成了。利用共軛焦顯微鏡以螢光共振能量轉移的技術就可以觀察到此融合蛋白的螢光性質。 我們利用實驗室之前發展的細胞轉染技術，可以同時轉染兩個不同的質體進入哺乳類細胞中表達，並且具有相當高的轉染效率，將蛋白酶及其特殊受質序列蛋白質共同轉染於人類胚胎腎臟細胞中，當我們增加蛋白酶的量時，可同時觀察到其特殊受質序列蛋白質中螢光共振能量轉移的訊號減弱甚至消失。因為是利用在活細胞中表達蛋白酶，我們可以利用此平台來驗證蛋白酶抑制劑在活細胞中的作用，尤其是那些以純化的蛋白酶所進行的抑制劑研究或電腦模擬所得的抑制劑在活細胞中的作用。對於愛滋病毒的蛋白酶，有報告指出pepstatin A具有抑制純化的蛋白酶活性的能力，不過在我們本次的論文中，以活細胞的平台，卻得不到相同的抑制效果。 最後，我們認為這個利用螢光共振能量轉移的技術在活細胞中研究蛋白酶活性的平台的確具有其可行性，並且它也適合用來決定受質的專一性，不同突變蛋白酶的酵素活性及測試突變蛋白酶對抑制劑的反應。最終，它可以很方便的轉換到高產能篩檢的模式，以進行蛋白酶抑制劑的大規模篩選。

Proteolysis is a process crucial to the normal turnover of proteins in the cell. Proteases are not only involved in protein destruction, but also in protein activation and numerous cellular processes. In addition, many viruses use cellular or viral proteases for various purposes. Consequently, specific protease inhibitors are attractive drug candidates. Although there were different assays used during recent years to assess protease activities, their actions were difficult to determine under physiological conditions. In this thesis, we tried to setup a cell-based assay which determined protease activity using fluorescence resonance energy transfer (FRET). We first constructed YFP-CFP fusion protein which contained multiple cloning sites (MCS) between two fluorescent proteins. Specific protease substrate sequences were introduced in-frame into the multiple cloning sites. A specific protease FRET probe was generated. Its FRET signal was readily detected by confocal microscopy. Utilizing a mammalian cell transfection method named surfection developed by our laboratory, we can cotransfect two plasmid simultaneously into mammalian cells with high transfection efficiency. Protease with its specific FRET probe were cotransfected into 293T cells with high efficiency. With increasing protease amount, the FRET signal was apparently decreasing. For its cell-based character, we used this assay for determining inhibition ability of cell free assay proved protease inhibitor. For HIV protease, pepstatin A had been shown to be an adequate inhibitor in cell free assay. But completely losing its inhibition to HIV protease in cell-based assay was noted in this thesis. In conclusion, this assay was capable of determining protease activity in cells. It was also suitable for determining substrate specificity or the different mutant proteases activity in cells. After all, it could be used for determining protease inhibitor and had the ability to convert into high throughput format for protease inhibitor screening.