Abstract：
Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.
摘要：
Toll样受体5（TLR5）响应鞭毛蛋白的单体形式并诱导MyD88依赖的信号通路，激活促炎转录因子如NF-κB和随后的细胞因子的诱导。另一方面,HMGB1是一种高度保守的非组蛋白染色体蛋白，可与TLR5相互作用并激活TLR5。本工作旨在使用全球分子对接平台，基于TLR5表面的结构知识，设计和表征源自SalmosalarHMGB1酸性尾部的TLR5激动剂肽。基于对接评分函数和预测的复合物的理论结合亲和力,过滤来自每个算法的在TLR5胞外域模型上复合的肽结合姿势。记录选择用于合成的每种肽的圆二色性光谱。只有内在无序的肽(6W，11W,和SsOri）被选择用于实验功能测定。通过NF-κB活化测定进行肽的功能表征，RT-qPCR基因表达测定,SHK-1细胞中的双眼皮次体和沙门氏菌攻击。6W和11W的肽增进了p65的核翻译和磷酸化。此外,肽诱导TLR5通路激活相关基因的表达,促炎和抗炎反应，以及T淋巴细胞向TH1，TH17和TH2等表型的分化和激活。最后,结果表明，11W肽保护免疫细胞免受沙门氏菌感染。总的来说,结果表明，新的肽作为潜在的免疫刺激剂的鲑鱼的有用性。
