IKBKB免疫缺陷病Y395H突变影响IKKβ蛋白磷酸化和免疫功能的机制研究

Deficiency of Innate and Acquired Immunity caused by IKBKB Mutation is a special and rare type of combined immunodeficiency disease, with severe immune function damage caused by IKKβ deficiency. In our preliminary data, we found that this patient had a homozygous point mutation that resulted in the substitution of histidine for tyrosine at position 395 (Y395H) and IKKβ protein was prone to degrate in patient and the mutant cell line. The immunity functions were impaired in this patient. These findings lead to the central hypothesis of the present study: IKKβ protein 395 amino acid site is an important phosphorylation site for IKKβ activation. The substitution of histidine for tyrosine at position 395 amino acid leads to the increased degradation rate of IKKβ protein resulted in affecting the canonical NF-κB activation pathway, which influence the adaptive immunity function consequently. In this study, we will utilize well established knock in cell model and mutant mice model to prove that the substitution of amino acid affects the degradation rate of IKKβ protein resulted in regulating NF-κB signal pathway. Through the research on hypothesis, we will add new content to the signal transduction pathway，but also the potential therapeutic target for anti-inflammation and anti-tumor therapy.

IKBKB基因缺陷导致一种罕见的、免疫表型十分特殊的重症联合免疫缺陷病，其基因突变可导致免疫功能严重受损。我们前期研究发现一患儿于IKKβ第395氨基酸位点纯合错义突变，由酪氨酸突变为组氨酸。对患儿PBMC及突变细胞系进行检测发现，突变后的IKKβ蛋白会迅速降解。患儿的免疫表型分析提示淋巴细胞功能受损。由此我们提出科学假设:IKKβ第395位酪氨酸为蛋白重要磷酸化功能位点，该位点突变将影响IKKβ蛋白活化表达及降解，从而影响NF-κB经典活化途径，使淋巴细胞免疫功能受损。本研究将利用成熟Knock in细胞模型和突变小鼠模型证实上述科学假设，阐明IKKβ395位点为重要的蛋白磷酸化位点，影响IKKβ蛋白活化表达，对NF-κB信号通路进行调控。该假设的验证不仅将为这一信号传导途径增添崭新的调控内容，也可能为感染、肿瘤等相关疾病提供潜在治疗靶点。

IKBKB基因缺陷导致一种罕见的、免疫表型十分特殊的重症联合免疫缺陷病，其基因突变可导致免疫功能严重受损。我们前期研究发现一患儿于IKKβ第395氨基酸位点纯合错义突变，由酪氨酸突变为组氨酸。对患儿PBMC及突变细胞系进行检测发现，突变后的IKKβ蛋白会迅速降解。患儿的免疫表型分析提示淋巴细胞功能受损。由此我们提出科学假设:IKKβ第395位酪氨酸为蛋白重要磷酸化功能位点，该位点突变将影响IKKβ蛋白活化表达及降解，从而影响NF-κB经典活化途径，使淋巴细胞免疫功能受损。.首先，我们通过构建IKKβY395H、IKKβY395E、IKKβS177A、IKKβS177E细胞系，使用DMSO或PP2处理细胞，检测发现395位酪氨酸产生突变后酪氨酸磷酸化IKKβ表达受损，并进一步使用TAK1抑制剂NG25和5Z-7-oxozeaenol对细胞进行刺激，检测发现酪氨酸磷酸化IKKβ表达水平下降，证明IKKβ395位点酪氨酸为该蛋白重要的磷酸化位点，其蛋白的稳定性依赖于TAK1激酶的激活。其次，通过体外细胞实验我们证实IKKβ395位的酪氨酸产生突变会加速IKKβ蛋白降解，但不会影响IKK复合物其他亚单位的正常表达。再次，我们的实验结果证明IKKβ蛋白Y395H突变并不影响NF-κB转录活性，但会影响NF-κB与其相关的DNA结合序列的相互作用；下调IL-1β、IL-6、IFN-γ、TNF-α、IL-10及IL-12等受NF-κB调节的炎症因子mRNA表达水平；使淋巴细胞免疫功能不同程度受损，尤其是使调节性Ｔ细胞的表达水平降低。.后续我们计划积极从IKKβ蛋白降解，如通过何种途径降解，具体的降解过程，有无特异的降解酶及降解酶抑制剂等进行严谨的实验设计，对该方面进行深入的探讨和研究，并期待能为感染性疾病及肿瘤疾病提供靶向治疗制药的理论及实践依据。