LPS对人CIAS1基因表达的影响及Cryopyrin结构与功能分析

发布时间：2018-06-29 15:53

本文选题：免疫识别 + CIAS1　；参考：《暨南大学》2006年硕士论文

【摘要】：固有免疫是多细胞生物抗御微生物和抗肿瘤等的第一道防线，固有免疫中感知病原的模式识别受体能识别微生物结构中常有的保守的病原体相关分子模式。在这些病原模式识别分子中，Toll样受体识别细胞外的病原体信息。胞浆中新发现的CARTERPILLER蛋白家族可能参与了对细胞内病原体及其产物的识别。由CIAS1基因编码的蛋白cryopyrin／NALP3／CIAS1／PYPAF1为CARTERPILLER蛋白NALP亚族成员，其基因突变可引起三种自发性炎症性疾病，分别为MWS，FCAS和CINCA／NOMID。由于cryopyrin同时存在PYRIN蛋白的PYD结构域与NOD蛋白的NACHT和LRR结构域，我们推测与自发性炎症性疾病有关的cryopyrin是重要的细胞内固有免疫相关的胞浆蛋白之一。但cryopyrin如何在细胞内被激活以及它又是如何触发免疫识别反应目前还不清楚。本研究采用分子生物学与理论计算相结合的方式，探讨cryopyrin在炎症发病机制中的作用及其各结构域在细胞内病原相关分子模式识别及信号级联反应中的作用。本实验用RT-PCR扩增正常人白细胞中CIAS1基因的NACHT区，观察不同时间及不同浓度LPS刺激对CIAS1的mRNA表达丰度的影响。另一方面，使用包括3D-PSSM，SPDBV，，Rasmol，WHAT IF，GRAMM和ExPASy等生物数据库资源和计算工具，对cryopyrin各结构域的三维结构进行模建，寻找它们在识别细胞内病原相关分子模式信号传导中的结构与功能的联系，并在结构分析的基础上初步探讨cryopyrin致病性突变(多分布在NACHT结构域)导致结构与功能变化的可能分子机制。 RT-PCR结果显示100μg／mL LPS刺激对CIAS1的mRNAL达呈现先降低后升高的模式。不同浓度LPS刺激2h，CIAS1 mRNA增高呈剂量依赖性(P＜0.01)。序列分析结果显示CIAS1基因产物cryopyrin的NACHT结构域存在能与ATP和Mg~(2+)结合的Walker A和Walker B基序。该基因的主要致病突变位点在结构上与此基序有密切关系。在LRRs区存在与Ca~(2+)和多糖结合的位点。NACHT结构域模型CASTp口袋分析表明：位于NACHT结构域中，正好在walker A基序和walker B基序的活性中心部位有一口袋，是磷酸腺苷ATP和Mg~(2+)结合及催化的部位。利用模型对接显示了该口袋结构与磷酸腺苷和Mg~(2+)的结合状态，表明存在该结构域与腺苷结合的互补性，与ATP结合的状态涉及的折叠变化目
[Abstract]:Innate immunity is the first line of defense against microorganisms and tumors in multicellular organisms. The pattern recognition receptors in innate immunity can recognize conserved pathogen-related molecular patterns in microbial structures. Toll-like receptors recognize extracellular pathogen information in these pathogenetic pattern recognition molecules. The newly discovered Cartel PILLER protein family in cytoplasm may be involved in the recognition of intracellular pathogens and their products. Cryopyrin / NALP3 / CIAS1 / PYPAF1, encoded by CIAS1 gene, is a member of CARTERPILLER protein NALP subfamily. Its gene mutation can cause three kinds of spontaneous inflammatory diseases, namely MWSFCAS and CINCAP / NOMID. Since the PYD domain of PYRIN protein and the NACHT and LRR domain of nod protein exist in cryopyrin, we speculate that cryopyrin associated with spontaneous inflammatory disease is one of the important cytoplasmic proteins associated with innate cellular immunity. But how cryopyrin is activated in cells and how it triggers immune recognition is unclear. In this study, molecular biology and theoretical calculations were used to investigate the role of cryopyrin in the pathogenesis of inflammation and the role of various domains in intracellular pathogen-related molecular pattern recognition and signal cascade reaction. The NACHT region of CIAS1 gene in normal human leukocytes was amplified by RT-PCR and the effects of LPS stimulation at different time and concentration on the mRNA expression abundance of CIAS1 were observed. On the other hand, using biological database resources and computing tools, such as 3D-PSSMN SPDBVN Rasmol-WHAT Ram and ExPASy, the three-dimensional structures of each domain of cryopyrin were modeled to find the relationship between their structures and functions in identifying the signal transduction of pathogen-associated molecular patterns in cells. On the basis of structural analysis, the possible molecular mechanism of structural and functional changes caused by cryopyrin pathogenicity mutations (mostly distributed in the NACHT domain) was preliminarily discussed. RT-PCR results showed that 100 渭 g / mL LPS stimulated CIAS1 mRNAL reached the first decreased and then increased. The increase of CIAS1 mRNA at different concentrations of LPS was dose-dependent (P < 0.01). Sequence analysis showed that the NACHT domain of CIAS1 gene product cryopyrin had Walker A and Walker B motifs that could bind to ATP and Mg2. The main pathogenicity mutation site of this gene is closely related to this motif in structure. In the LRRs region, there is a site binding to Ca ~ (2) and polysaccharides. The CASTP pocket analysis of NACHT domain model shows that there is a pocket in the active center of walker A motif and walker B motif, which is located in the NACHT domain. Adenosine phosphate ATP and mg ~ (2) binding and catalytic site. The binding states of the pocket structure to adenosine phosphate and mg ~ (2) were demonstrated by using the model docking, indicating the complementarity between the domain and adenosine, and the folding change of ATP binding state.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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