微管相关蛋白4(MAP4)在促微管聚合药物早期激活NF-κB作用及机制研究

Microtubules play an important role in the inflammatory reaction, promoting the microtubule polymerization drugs (taxol, limulus anti-polysaccharide derived peptide CLP - 19) in the early effect (1 h) can activate the NF-kappa B, but its mechanism is yet to be studied. Our preliminary study found that this function is related to activation TNFR intracellular domain structure and up regulated the TRAF2 ubiquitin levels. Both taxol and CLP-19 can boost phosphorylation levels of microtubule associated protein 4 (MAP4) Ser768 at the early stage, but phosphorylation level of NF-kappa B was the cell significantly reduced in which knockdown by siRNA of MAP4. Given the phosphorylation is an important mechanism for regulating MAP4, we put forward the " drugs which can promote the microtubule protein aggregation at the early stage can up regulate MAP4 kinase phosphorylation, further activate TNFR intracellular structure domain, so as to activate the NF-kappa B" hypothesis.This study intends to clarify the change of MAP4 phosphorylation site by the drug which can promoting the microtubule protein aggregation, the change MAP4 expression impact on the phosphorylation of NF-kappa B, and the interaction between MAP and TRAF2, the mRNA and protein change induced by the promote microtubule polymerization drugs at the early stage. And this study eventually clarify the MAP4 phosphorylation change induced by promote the microtubule polymerization drugs at the early stage, and furher under the action of drugs early MAP4 phosphorylation change and its role in the nf-kappa B activation, and lay a foundation of MAP4 phosphorylation sites of new drug design.

微管在炎症反应中起着重要作用，促微管聚合药物（紫杉醇、鲎抗内毒素因子模拟肽CLP-19）在作用早期（1h）可激活NF-κB，然其机制尚待研究。我们前期研究发现该作用与激活TNFR胞内结构域，上调TRAF2泛素化水平相关；而紫杉醇、CLP-19早期作用后微管相关蛋白4（MAP4）Ser768磷酸化水平上调，而沉默MAP4后激活NF-κB作用显著降低。鉴于磷酸化是调节MAP4的重要机制，我们提出"促微管蛋白药物早期使MAP4激酶磷酸化，进一步激活TNFR胞内结构域，激活NF-κB"的假设。本研究拟明确促微管聚合药物早期对MAP4 磷酸化位点变化、MAP4表达改变后对NF-κB通路的影响；MAP4与TRAF2交互作用及促微管聚合药物早期作用下的基因、蛋白水平的影响。最终明确促微管聚合药物作用下早期MAP4磷酸化改变及其在NF-κB激活中的作用，并为针对MAP4磷酸化位点新药设计奠定基础。

微管在炎症反应中起着重要作用，促微管聚合药物（紫杉醇、鲎抗内毒素因子模拟肽CLP-19）在作用早期可激活NF-κB，然其机制尚待研究。在期研究发现该作用与激活TNFR胞内结构域，上调TRAF2泛素化水平相关；而紫杉醇、CLP-19早期作用后微管相关蛋白4（MAP4）Ser768磷酸化水平上调，而沉默MAP4后激活NF-κB作用显著降低。本研究旨在明确促微管聚合药物早期对MAP4 磷酸化位点变化、MAP4表达改变后对NF-κB通路的影响，以为促微管聚合药物作用下早期MAP4磷酸化改变及其在NF-κB激活中的作用，并为针对MAP4磷酸化位点新药设计奠定基础。.课题首先明确了促微管聚合药物紫杉醇、CLP-19对微管聚合剂量因素，发现在低浓度条件下，紫杉醇、CLP-19和秋水仙碱对微管蛋白无明显影响，然而在有致炎因子条件下，紫杉醇、CLP-19可抑制致炎因子对微管解聚，该作用具有显著的时间剂量效应。并对CLP-19与RAW 264.7细胞特异性结合蛋白的鉴定和作用特征，结果发现，CLP-19可与微管异二聚体结合，并通过多肽亲和色谱发现CLP-19结合生物信息学计算机模拟分子对接实验显示CLP-19可能主要结合在α、β微管异二聚体的凹槽位置，并和α/β微管蛋白均可发生键合。课题进一步研究了促微管聚合药物紫杉醇、CLP-19对微管聚合的时间因素，在胞外，CLP-19可促进微管聚合，在不同剂量下，对微管的通透性有显著影响，在LPS、TNF-a 处理不同时间后，MAP4（ser 696,ser 787）的磷酸化均显著增强，该作用呈显著的时间效应。以紫杉醇，CLP-19作用后，对LPS所致MAP4的磷酸化有显著抑制作用，而秋水仙碱作用后，对MAP4磷酸化有加强作用。CLP-19与其它药物合用，可显著降低其它药物对细菌的MIC，推测该作用与CLP-19和细菌微管作用相关。课题发表文章4篇，其中SCI文章3篇，CSCD文章1篇，申请发明专利1项。