LAPF调控抗病毒天然免疫效应与机制研究

Type I interferon is critical for anti-viral function by inducing interferon-stimulating gene. LAPF （Lysosome-associated Apoptosis-inducing protein containing PH and FYVE domains）was first reported by our lab through large scale sequencing and it could promote apoptosis of fibroblasts. We found that LAPF expression significantly decreased in macrophage from heterozygote of LAPF knockout mice, LAPF bound Akt vigorously and promoted its activation, LAPF also increased the reporter gene of IFN-β, which indicated that LAPF may increase IFN-β production and anti-viral function by promoting Akt activation. The proposal plan to breed the homozygote LAPF knockout mice, systemically investigate the function of LAPF on type I interferon induction and the inhibition of virus replication and key signal transduction pathway in vivo and in vitro. The proposal will explore the key post-translational modification types and amino acid sites of LAPF activity regulation， the key interaction protein by constructing PH and FYVE function lost mutant and followed mass-spectrum detecting the immunoprecipitation production. The accomplishment of proposal will provide new lights on molecular mechanism of anti-viral innate immunity and novel anti-viral drug target.

I型干扰素在对抗病毒感染中发挥至关重要的作用。 溶酶体相关分子LAPF是由本实验室通过大规模测序独立发现的、能够促进成纤维细胞和肿瘤细胞凋亡的新分子，我们预实验发现LAPF敲除杂合子小鼠的巨噬细胞中LAPF表达明显降低，杂合子巨噬细胞应对水泡性口炎病毒感染（VSV）时产生IFN-β显著减少；LAPF促进IFN-β报告基因活化、结合并促进Akt活化，提示LAPF可能通过活化Akt促进IFN-β产生和增强抗病毒效应。本项目拟运用首次报道的LAPF缺陷小鼠，体内体外系统研究LAPF在巨噬细胞或气管上皮细胞中促进干扰素产生和抑制病毒复制效应与作用环节；通过免疫共沉淀后质谱鉴定技术，研究调控LAPF活化的关键翻译后修饰类型及其位点、LAPF关键相互作用分子。为抗病毒天然免疫关键分子机制研究提供新线索和抗病毒药物设计提供新靶点。

巨噬细胞介导的病原体内吞和随后的溶酶体降解机制是宿主对抗病原体感染的重要机制。我们研究发现早期内体定位的LAPF可以促进巨噬细胞吞噬细菌，促进细菌感染引起的炎症反应。促进病毒感染引起的IFN产生，促进抗病毒天然免疫反应。体内实验证实LAPF小鼠感染E. Coli后，虽然炎症反应降低，但是细菌在体内增殖播散更严重，LAPF小鼠更容易死亡。细胞水平研究证实LAPF缺陷的巨噬细胞吞噬E.Coli和S. Aureus减少，炎症反应减少。LAPF缺陷的巨噬细胞吞噬E.Coli和S. Aureus后，杀灭细菌能力减少。机制研究表面酪氨酸磷酸激酶可以磷酸化LAPF和Caveolin，促进LAPF和Caveolin复合体形成；268位酪氨酸磷酸化的LAPF可以促进Caveolin1/2聚集。Src抑制剂取消LAPF和Caveolin复合体形成，Src抑制剂体内注射导致小鼠感染E. Coli更容易死亡，体内细菌荷载量更多。