Talin 1片段化对HIV入侵和突触形成的影响及分子机制研究

Talin 1 (a cytoskeletal protein) takes part in HIV invasion and synapse development, however, the detail mechanism is unknown. We found talin 1 could be broken into 38KDa fragment through HIV infection.This fragment is negatively related to HIV load, further, in vitro study showed this fragment could decrease HIV infection. This fragment might be produced by HIV-1 protease. One of the binding domains of integrin is left in this fragment. Herein, we speculate that the fragment of talin-1 regulate integrin activity and signal pathway to affect HIV invasion, membrane fusion and immune synapse development. In this program, we study the interaction between HIV and talin 1 fragment and its affection to integrin activities and signal pathway through HIV infection cell models, iTRAQ labeled plasma membrane proteomics、molecular cytobiology, etc. At last, the proteins interacted with talin 1 fragment were verified in clinical samples to find the relationship between their expression and HIV loading or talin 1 fragment through MRM method based on mass spectrometry. In all, a "molecular network-cell-individual" study will be performed. Through this program, we will discover the reason that HIV induce talin 1 fragmentaion, the method that talin 1 fragment regulate HIV infection through cell signal pathway, and dicover the mechanism of HIV inducing talin 1 fragmentation and fragment decrease HIV infection and negatively related to HIV loading. Our study will offer new clues to the mechanism of HIV invasion and synapse development.

细胞骨架踝蛋白（talin 1）参与HIV入侵和突触形成过程，但具体机制不明。我们发现HIV感染诱导踝蛋白1断裂成38KDa片段，该片段与病毒载量负相关，且体外研究表明片段抑制HIV感染。该片断可能被HIV蛋白酶切割且部分保留了整合素（integrin）的结合位点。故推测踝蛋白1片段调节整合素网络，从而影响HIV入侵及通过膜融合和突触形成进行传播的过程。本研究用病毒感染细胞模型、iTRAQ标记的细胞质膜蛋白质组学等方法研究片段对HIV入侵、突触形成和膜融合的影响；片断对整合素活性及其信号通路的调控；临床验证踝蛋白1片段相互作用蛋白质与病毒载量的关系。从分子网络－细胞－个体水平探讨HIV诱导踝蛋白1片断化的原因，片段如何通过影响细胞信号通路来调控HIV感染，揭示病毒感染诱导踝蛋白 1片段化，片段抑制病毒感染以及与病毒载量负相关的分子机制，为HIV入侵和突触形成分子机制研究提供新线索。

艾滋病给我国政治、经济带来重大影响。细胞骨架踝蛋白（talin 1）参与HIV 入侵和突触形成过程，但具体机制不明。我们发现HIV 感染诱导踝蛋白1 断裂成38KDa 片段，该片段与病毒载量负相关，且体外研究表明片段抑制HIV 感染。该片断可能被HIV 蛋白酶切割且部分保留了整合素（integrin）的结合位点。故推测踝蛋白1 片段调节整合素网络，从而影响HIV 入侵及通过膜融合和突触形成进行传播的过程。本研究构建了过表达talin 1质粒的瞬时转染TZM-b1细胞，38KDa talin 1片段、talin 1头部和ShRNA敲低talin 1的稳转TZM-b1细胞株。用HIV假病毒感染以上4种细胞，发现过表达talin 1片段可以抑制HIV假病毒的感染，而敲除talin 1片段会增加HIV假病毒的感染率。这些结果表明，talin 1片段具有抗HIV感染的作用。随后利用iTRAQ标记的蛋白质组学技术研究了talin 1片段过表达对HIV假病毒感染TZM-b1细胞过程中蛋白质表达的影响，发现了7个差异蛋白质，如Annexin A1和Zyxin在过表达Talin 1片段的细胞中上调。研究了HIV整合细胞U1，A7及其对应的对照细胞系中细胞质膜蛋白质的变化，发现了CAPG， Integrin等受HIV调控的蛋白质,生物信息学分析发现talin 1-integrin等形成相互作用的蛋白质网络。在HIV感染的临床样品中分析了CAPG的表达。本研究为HIV 入侵和突触形成分子机制研究提供新线索。