Effect of Arginine Side Chain Length on the Biological Functions of Tat-Derived Peptides
Date Issued
2013
Date
2013
Author(s)
Wu, Cheng-Hsun
Abstract
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.
Subjects
非自然界胺基酸
Tat衍生胜肽
辨識核糖核酸
細胞穿透胜肽
中孔洞二氧化矽奈米粒子輸送
SDGs
Type
thesis
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