基于CRISPR/Caspase9基因编辑技术研究狼疮致病基因IFIT4(RIG-G)蛋白乙酰化修饰机制及其在B细胞免疫耐受、抗细胞增殖中的功能作用和分子机制

In systemic lupus erythematosus (SLE), B cell immune tolerance is abnormal, producing a large number of auto-antibodies which play a pathogenic role. The expression of IFIT4 (also known as RIG-G) in peripheral blood of human (especially in SLE patients) is highly correlated with the disease, suggesting its important pathogenic role, but its function is rarely known. Protein acetylation is one of the prerequisite factors for the function of many proteins. Our preliminary experiments showed that IFIT4 protein can be modified by acetylation. The key site is k373, whose function and pathogenicity involved with lupus are unclear. Therefore, this project intends to investigate the function and molecular mechanism of IFIT4-K373 acetylated protein in B cell immune tolerance and anti-cell proliferation in order to clarify its mechanism in the pathogenesis of lupus. After elucidating the mechanism of IFIT4 acetylation, we used CRISPR /Caspase9 technology to prepare KR (Lysine K mutated into Arginine R) or KQ (Lysine K mutated into Glutamine Q) point mutation at K373 site to creat KR acetylation defects, or KQ simulated point mutation in mice, and IFIT4 knockout mice; then hybridized those mice with spontaneous lupus mice to identify their impact on lupus. Bone marrow transplantation for re-construction by using recombinant B cell tolerance mouse model and in vitro clonal clearance related with naïve B cell model to identify its role in B cell immune tolerance; Finally, the use of gene chip, mass spectrometry, co-immunolocalization, point mutation, SiRNA, FCM and other technologies to identify its downstream genes and interacting proteins to clarify the molecular mechanisms.

系统性红斑狼疮（SLE）中，B细胞免疫耐受异常、产生大量自身抗体发挥致病作用。IFIT4 (又称RIG-G)在人(尤其在SLE患者)外周血B细胞特异性高表达，提示重要致病作用，但功能研究极少。蛋白乙酰化修饰是多种蛋白发挥功能的前提，我们预实验发现IFIT4蛋白可被乙酰化修饰、关键位点为K373，其功能不清。因此，本项目拟探讨IFIT4-K373乙酰化蛋白在B细胞免疫耐受中的功能机制。利用CRISPR/Caspase9技术制备K373位点的KR（赖氨酸K突变成精氨酸R）、或KQ（赖氨酸K突变成谷氨酰胺Q）的乙酰化缺陷、或模拟点突变小鼠、及IFIT4基因敲除鼠，与自发性狼疮鼠杂交鉴定其对狼疮发生的影响；骨髓回输重建B细胞耐受小鼠模型、和体外克隆清除的Naïve B细胞模型等鉴定其对B细胞免疫耐受的作用；RNA-seq、质谱、免疫共定位、SiRNA等技术鉴定其下游基因和相互作用蛋白等分子机制。

目标：本预实验发现 IFIT3蛋白在系统性红斑狼疮（SLE）明显B细胞高表达，但其功能不清。本项目拟探讨IFIT3及其乙酰化修饰在SLE的B细胞分化中的作用及分子机制。方法： 蛋白质谱和点突变鉴定IFIT3的乙酰化位点。通过 在细胞和MRL/lpr狼疮易发小鼠中，构建IFIT3及其IFIT3乙酰化位点K373位点的过表达和Crispr/CAS9的IFIT3的敲除的细胞和小鼠，鉴定小鼠狼疮表型和骨髓、脾、骨髓不同发育阶段的B细胞和浆细胞分化表面标记物、相关信号通路分子。结果。SLE的B细胞IFIT3高表达与抗ds-DNA抗体和疾病活动度正相关。发现IFIT3有8个乙酰化位点，K373是最关键的乙酰化位点，对IFIT3蛋白稳定性发挥作用。IFIT3过表达的MLR/lpr 小鼠促进了IgG1抗体的产生、浆细胞分化（CD138+的频率更高），增加了浆细胞特征Blimp1和IRF4的表达，并减少了Bcl6。 MRL/lpr狼疮小鼠的IFIT3 KO产生了相反的效果，改善了狼疮肾炎表型。 IFIT3通过与TBK1交互，导致激活 TBK1和NF-κB家庭成员p65/RelA，促进了浆细胞的分化和自身抗体产生。然而IFIT3的K373位点突变小鼠未影响B细胞分化。结论。 在SLE高表达的IFIT3通过激活TBK1和p65而在B细胞分化和抗体产生中发挥重要作用 ，IFIT3的K373乙酰化位点控制IFIT3蛋白稳定性。拟投稿2篇，其一投稿Arthritis and Rheumatism (影响因子10分-14分)