穿透性胜肽媒介蛋白質進入細胞之機制探討

Molecular Analysis of Cell Penetrating Peptide-Mediated Transport of Cargo Proteins

劉昶辰(Chang-Chen Liu)

指導教授 ： 張富雄

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00910

摘要

到目前為止，有許多文獻報導細胞穿透胜肽(cell penetrating peptide)或蛋白轉位領域(protein transduction domain)在遞送許多物質進入細胞，但是科學家們對於穿透性胜肽轉位進入細胞的機轉所知不多。由於穿透性胜肽本身為一個高極性的分子，所以可以快速、有效率的直接穿透細胞膜上的脂雙層(lipid bilayer)，攜帶物質到達細胞質以及細胞核，所以我們為了想要了解穿膜的機轉，利用不同功能區塊的組合，建構了四個融合蛋白來驗證。包含streptavidin的核心區域(ST)、protein A的BC domain(PA)以及Tat domain。利用這些功能性的區塊，可以協助我們更加的了解穿透性胜肽在蛋白轉位作用當中所扮演的角色。利用流式細胞儀以及雷射共軛焦螢光顯微鏡研究是否Tat胜肽可以穿透不同種類的細胞以及不同時間內的作用。結果顯示Tat-PA-IgG以及Tat-PAST-IgG於短短的30分鐘內，在細胞中就有吞入螢光的訊號產生，隨著時間的增加，有越來越多的細胞其內含有螢光訊號且在6小時達到飽和。這樣的結果，到達一個門檻劑量之後，再隨著劑量的增高，對於Tat轉位效率也沒有顯著的增強。利用ATP抑制劑處理細胞，也沒有發現有抑制的效果，這讓我們知道Tat進出細胞是與能量無關的(energy-independent)。利用caveolae抑制劑nystatin，亦沒有顯著的抑制。最後利用macropinocytosis抑制劑amiloride(Na+/K+ pump inhibitor)處理細胞之後，對於穿透的效率也沒有影響。但是，在4oC的條件下，卻發現有抑制Tat融合蛋白進出細胞的效果，所以推測Tat蛋白進出細胞可能是藉由endocytosis的機轉。利用lipid raft的抑制劑methyl-βCD，也可以發現有抑制的效果。這也證實了我們所建構的Tat融合蛋白攜帶貨物遞送的路徑，可能是藉由lipid raft所傳導的內噬機轉。因此，本篇論文證明了Tat融合蛋白進入細胞的機制，其所衍生出來的多功能穿透性蛋白亦是一個相當有價值的工具平台，除了可以用來遞送高分子量以及一些特殊的物質進入各式各樣的細胞之外，同時也提供了一個快速純化多功能融合蛋白的技術平台。相信對於未來在蛋白質治療的研究上，會有相當大的助益。

關鍵字

穿透性胜肽蛋白轉位作用

並列摘要

Delivery of macromolecules mediated by cell penetrating peptides (CPPs) or protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Because of CPPs are the high polar molecules, its can fast and efficiency direct translocation the lipid bilayer in the cell surface.
Internalization of proteins into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of cargos into cells using the TAT-fused protein. This fusion protein consists of three functional domains, the protein transduction domain of HIV-1 TAT；the B、C domain of staphylococcal protein A，which has an ability to bind to the IgG；and the core region of the streptavidin，which has an ability to bind to the biotinylation molecules. The TAT–PAST fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence labeled IgG with the TAT–PAST fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. we demonstrate that the entry of Tat-PAST peptide fusion protein into HeLa cells is serum-dependent、ATP-independent and temperature dependent, indicating the involvement of endocytosis. Specific inhibitors of lipid- raft-dependent endocytosis partially inhibit Tat-PAST fusion protein uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway and macropinocytosis pathway are not essential for the uptake of Tat-PAST fusion protein. It proves to be a useful technique for screening functional proteins and peptides from cell or cell-free lysate without purification. Based on this possibility, we have developed a novel method for the internalization of antibodies using the TAT-fused protein.

並列關鍵字

cell penetrating peptide ； protein transduction ； Tat

參考文獻

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