TYK2致病变异通过影响JAK-STAT信号转导通路抑制麻风免疫应答的分子机制研究

TYK2 has been identified as one of susceptibility genes of leprosy previously. Next generation sequencing has been employed to determine the rare variant rs55882956 (G/A,p.R703W), which was located in the pseudokinase domain of TYK2. This domain did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein. Mutation of the domain ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling.In our previous study, the result of protein structural analysis suggests that the rs55882956 has a strong destabilizing effect on the protein structure, indicating that the rare variant may be involved in pathogenesis of leprosy via influence the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal transduction pathway, but the mechanism remains unclear. To figure out that question, the rare variant rs55882956 (p.R703W) have been validated to be causal variant in 1,000 leprosy cases and 1,000 health controls by genotyping analysis. Furthermore, transcriptome sequencing,qPCR, immunochemistry, Western Blot will be conducted to clarify how the causal variant affects the function of TYK2. The mechanism of causal variant will be explored using gene knock-down Zebrafish. The phosphorylation of TYK2 and STAT will be detected in vitro methods. This study will be greatly advancing the biological understanding of leprosy as well as other mycobacteria infectious diseases.

课题组前期发现TYK2为麻风易感基因，深度测序显示其编码区域变异位点rs55882956与疾病的相关性最强。rs55882956位于TYK2蛋白的假激酶功能区域，蛋白结构分析发现该变异影响TYK2蛋白结构稳定性，提示其可能通过妨碍JAK-STAT信号转导,进而影响巨噬细胞、树突状细胞、NK细胞等对病原微生物的识别及杀伤，从而导致疾病的发生,目前尚缺乏实验证实。课题组已对测序所得变异位点进一步验证并明确为致病变异。后续课题组拟利用转录组测序、qPCR、免疫组化、免疫印迹等技术阐述致病变异对TYK2基因功能的影响；而后利用吗啉代反义寡核苷酸Knock-down斑马鱼探讨致病变异的致病性；同时构建突变型细胞系，检测该变异位点对JAK-STAT信号通路蛋白磷酸化水平影响，以最终阐释麻风的发病机理，为疾病风险预测、疾病干预等累积基础。

背景：课题组前期发现TYK2为麻风易感基因，变异位点rs55882956与疾病的相关性最强。rs55882956位于TYK2蛋白的假激酶功能区域，蛋白结构分析发现该变异影响TYK2蛋白结构稳定性，提示其可能通过妨碍JAK-STAT信号转导,进而影响机体对病原微生物的识别及杀伤，导致疾病的发生,但目前尚缺乏实验证实。.研究内容：.1.研究TYK2基因和其免疫通路相关基因在麻风患者和健康对照皮肤组织中转录水平的表达差异：选取麻风病例27例（多菌型19例，少菌型8例）及健康志愿者18例的皮肤组织，进行RNA-SEQ实验；选取额外的患者11例（多菌型9例，少菌型2例）及5例健康志愿者皮肤进行QPCR实验；.2.研究TYK2蛋白在麻风患者和健康对照皮肤组织中是否有表达差异：选取10例（其中多菌型5例，少菌型5例）麻风患者及健康志愿者5例的皮肤蜡块组织进行免疫组化实验；.3.研究海鱼分枝杆菌感染小鼠巨噬细胞后TYK2转录水平的表达：用不同剂量的海鱼分枝杆菌刺激RAW264.7细胞，共培养，分别在不同时间点收集细胞，通过qPCR测定TYK2mRNA表达量；.4.研究海鱼分枝杆菌感染诱导为巨噬细胞后的人THP-1细胞后TYK2蛋白翻译水平的表达：用不同剂量的海鱼分枝杆菌刺激诱导为巨噬细胞的THP-1细胞，分别在不同时间点收集细胞通过western-blot测TYK2蛋白表达量。.结果：.1.RNA-SEQ实验、qPCR实验中麻风组织中TYK2转录水平TYK2mRNA表达量均明显高于正常组织（P<0.01），但TYK2表达量在多菌性（MB）和少菌型（PB）麻风患者组织中并无差异（P>0.05）；.2.免疫组化示麻风患者组织中TYK2蛋白表达量明显高于正常对照组织，且表达量在MP、BP中并没有明显差异（P<0.01）；.3.与TYK2通路相关的多个基因在麻风组织中的表达量明显高于正常对照组（P<0.01）;.4.海鱼分枝杆菌刺激RAW264.7细胞后TYK2mRNA表达量明显升高，且表达量与菌量成正相关；.5.海鱼分枝杆菌刺激诱导为巨噬细胞的THP-1细胞，TYK2蛋白表达量明显升高，且与刺激时间成正相关。.结论：本次实验首次报道基因TYK2与麻风的可能相关性，其可能是通过参与巨噬细胞对麻风分枝杆菌的吞噬作用参与到麻风的免疫反应过程。