精胺酸側鏈長短對Tat衍生胜肽之生物功能的影響

Abstract

精胺酸的側鏈長短對蛋白質結構穩定上的影響已有相當的結果，然而在生物功能方面的應用與影響仍需進一步的研究。在人類免疫缺乏病毒Tat蛋白質中的一段胜肽序列:Tat(47-57)同時具有與病毒TAR RNA結合及穿透細胞膜的能力，且此序列上的六個精胺酸對於TAR RNA辨認及穿透細胞膜相當重要。在此研究中將此胜肽序列作為模組來研究精胺酸側鏈長短對Tat衍生胜肽生物功能的影響。Tat衍生胜肽為將Tat(47-57)胜肽鏈上的六個精胺酸同時取代為Agh (側鏈較Arg多一個亞甲基)，或Agb (側鏈較Arg少一個亞甲基)。利用螢光各向異性度分析和膠體電泳偏移分析來研究Tat衍生胜肽與TAR RNA的解離常數。利用螢光顯微鏡觀察及流式細胞儀測量Tat衍生胜肽進入Jurkat細胞內部的效率。此外也以圓二色光譜儀偵測Tat衍生胜肽在與TAR RNA及仿細胞膜脂肪聚合物結合時其二級結構上的變化以求了解其機制。在AghTat的結果中將側鏈加長會降低與TAR RNA的專一性但增強進入細胞的能力。而將側鏈縮短時(AgbTat)會同時增加與TAR RNA的專一性及穿透細胞膜的能力。此結果顯示將側鏈縮短的AgbTat衍生胜肽或許可應用於藥物傳輸及人類免疫缺乏病毒治療。 為了更進一步發展出對人類免疫缺乏病毒TAR RNA有好的結合能力的Tat衍生胜肽。將Tat(47-57)模組上的六個精胺酸分別取代成Agh，Agb及Agp (側鏈較Arg少二個亞甲基)，藉膠體電泳偏移分析得知不同側鏈長短的Arg衍生物在不同位置上對TAR RNA專一性結合的影響。再將不同位置上具有最佳專一性的Arg衍生物重新組合成一組在不同位置具有不同側鏈長度的Tat衍生胜肽且具有對人類免疫缺乏病毒TAR RNA最好的專一性結合能力。此系列胜肽也同時增強進入細胞的能力，且在細胞缺乏ATP的條件下，胜肽仍可經由non-endocytosis機制進入細胞。由非自然界胺基酸所組成之胜肽鏈，具有抗胰蛋白酶(trypsin)水解之能力，此外此胜肽鏈也可在細胞內部維持24小時以上。利用含有HIV TAR RNA及luciferase基因修飾的Jurkat細胞證實此系列胜肽鏈，可有效抑制HIV TAR RNA基因段的轉錄。根據研究結果此系列胜肽鏈或許可應用於藥物傳輸及人類免疫缺乏病毒治療。 將此研究中所發現具有較好細胞膜穿透能力的Tat衍生胜肽與不同尺寸的中孔洞二氧化矽奈米粒子結合，研究精胺酸側鏈長短對不同尺寸奈米粒子輸送效率及機制的影響。利用流式細胞儀測量奈米粒子在不同環境下進入HeLa細胞內部的效率，再以共軛焦顯微鏡觀察其在細胞內部的分布以推測其機制。結果顯示中孔洞二氧化矽奈米粒子進入細胞的效率與機制與其粒子大小及表面修飾有關。另外在300奈米尺寸下，非自然界Tat衍生胜肽可有效幫助中孔洞二氧化矽奈米粒子進入細胞質。

The guanidinium bearing side chain of arginine (Arg) is unique and critical for various bioactivities. However, the functional role of the three hydrophobic methylenes linking the hydrophilic guanidinium group to the backbone of Arg remains unclear. As such, the effect of arginine side chain length on RNA recognition and cellular uptake has been investigated. The 11-amino acid basic region of HIV Tat protein (human immunodeficiency virus transactivator of transcription protein, residues 47-57) binds to the transactivator response element (TAR) RNA, and is responsible for cell penetration. The six arginines in the Tat(47-57) peptide were all simultaneously replaced by arginine analogs (S)-2-amino-6-guanidinohexanoic acid (Agh, 4 methylenes) or (S)-2-amino- 4-guanidinobutyric acid (Agb, 2 methylenes). These Tat-derived peptides were synthesized by solid phase peptide synthesis (SPPS). The binding specificity was determined by electrophoretic mobility shift assays (EMSA). The cellular uptake into Jurkat cells were determined by flow cytometry. Lengthening the Arg side chain length by one methylene to give AghTat greatly diminished specific binding of TAR RNA, with slightly increased cellular uptake. Surprisingly, shortening the Arg side chain length to give AgbTat enhanced both TAR RNA binding specificity and cellular uptake. As such, AgbTat may be useful for developing drug delivery vehicles or anti-HIV therapeutics. The effect of side chain length at the different positions of Tat(47-57) for binding TAR RNA was explored. For position 57, Arg and Agb exhibited similar binding specificity. For position 56, the TAR RNA binding specificity followed the trend Agb > Agp > Arg > Agh (Agp, side chain two methylenes shorter than Arg). For position 55, Arg and Agp exhibited similar binding specificity. For 53 position, the TAR RNA binding specificity followed the trend Agp > Agb ~ Arg > Agh. For positions 52 and 49, the TAR RNA binding specificity followed the trend Agp > Arg > Agb > Agh. The potentially most optimal Tat-derived peptide for specific binding to TAR RNA with Arg analogs of mixed side chain lengths was designed by combining the Arg analogs with the highest binding affinity at each position. The Tat-CX peptides exhibited higher TAR RNA binding specificity and cellular uptake compared to ArgTat. Furthermore, a portion of the cellular uptake for the Tat CX peptides apparently occurred via non-endocytotic pathways. The non-natural amino acids containing Tat-CX peptides exhibited high resistance to trypsin proteolysis and intracellular degradation. Importantly, most Tat-CX peptides inhibited Tat-dependent luciferase gene expression more than native ArgTat. These results suggest that the non-natural Tat-derived peptides (Tat-CX) may be effective peptide transporters for drug delivery application and anti-HIV therapeutics. Preliminary temperature dependence studies on cellular uptake of Tat-derived peptides suggested non-endocytotic uptake. Accordingly, analogous Tat-derived peptides (XaaTatC) were conjugated onto MSNs with different sizes to affect the cellular uptake efficiency of the MSNs. The uptake into HeLa cells were investigated by flow cytometry and confocal microscopy. Results showed size dependence in cellular uptake efficiency and mechanism of the MSNs. Furthermore, endosome trapping of the 300 nm MSN was circumvented by improving the efficiency of endosomal escape and even enhancing non-endocytotic pathways. Importantly, introducing cell-penetrating peptides containing non-natural amino acids analogs with varying side chain lengths should be a useful strategy for the design of nanoparticle surface ligands for biomedical applications.