酵母双杂交筛选NLRP3相互作用蛋白质

发布时间：2018-06-02 07:33

本文选题：固有免疫 + NLRP3　；参考：《中南大学》2013年硕士论文

【摘要】：摘要：在哺乳动物体内,存在多套免疫识别系统,通过维持机体与共生菌群的正常互动以及响应外来病原和刺激的免疫反应,从而维持组织稳态。与主要识别微生物来源病原相关分子模式的、存在于内吞体和细胞膜上的Toll-like receptors (TLRs)不同,定位于胞内的Nod-like receptors (NLRs)不仅识别胞内病原相关分子模式,也识别宿主来源的损伤相关分子模式,比如机体因胁迫、组织损伤和细胞死亡而产生的内源危险信号。 NLRs是重要的胞质内模式识别受体,研究发现其大多数成员能构成一个大的分子平台,如最初发现由NLRP1、ASC、caspase-1和caspase-5构成,这个大的复合物被定义为"inflammasome",即炎症小体,它被证明是促炎细胞因子IL-1β (Interleukin-1β)活化所必需的。之后,许多NLRs[2-5]家族和PYHINs家族的感受蛋白也相续被证明能在各种Damage-associated molecular patterns (DAMPs)和Pathogen-associated molecular patterns (PAMPs)刺激下形成功能性炎症小体。现在,炎症小体的提出已被公认为细胞内免疫监视系统里程碑式的发现。机体内,炎症小体广泛参与了抗微生物感染的先天性免疫反应,其中研究得最广泛、最深入的就是NLRP3(NLR family, pyrin domain containing3)炎症小体。基于NLRP3炎症小体的分子靶向治疗目前也正在研究当中。因此,深入发掘NLRP3信号通路分子调控机制,有可能为揭示炎症、免疫及疾病之间的相互关系提供重要依据,同时为某些疾病治疗提供新的靶点。近年来,通过对NLRP3炎症小体及其相互作用蛋白质的研究,增加了人们对NLRP3功能调控的认识。因此,为进一步探索关于炎症小体的信号调控机制及其生物学功能提供基础,本项研究通过酵母双杂交的技术手段筛选与NLRP3相互作用的蛋白质。以NLRP3三个基本结构域PYD、NACHT、LRR为诱饵,在人白细胞cDNA文库中筛选其相互作用蛋白质。经过酵母细胞内进一步验证,我们从87个初步阳性克隆中筛选出了4个与NLRP3相互作用的候选阳性克隆,即ECHS1(enoyl CoA hydratase, shortchain,1)、 UBE2I(ubiquitinconjugating enzyme E2I)、RPL34(ribosomal protein L34)以及PXN(paxillin)。进一步在真核细胞内验证了这些候选蛋白质与NLRP3的相互作用,免疫共沉淀实验(Co-IP)表明ECHS1能够结合NLRP3。本项研究发现NLRP3和ECHS1的相互作用可能是一个新的NLRP3调控因素,进一步深入研究有可能发现一个关于NLRP3功能调控的新机制。图15幅,表3个,参考文献58篇
[Abstract]:Abstract: in mammals, there are many sets of immune recognition systems to maintain the tissue homeostasis by maintaining the normal interaction between organism and symbiotic flora and the immune response to exogenous pathogens and stimuli. Different from the molecular patterns related to the identification of pathogenic pathogens from microorganisms, Toll-like receptors TLRs, which exist in endocysts and cell membranes, not only recognize intracellular pathogen-related molecular patterns, but also Nod-like receptors nlRs, which are located in cells. Host-derived damage-related molecular patterns such as endogenous danger signals from stress tissue damage and cell death are also identified. NLRs is an important intracytoplasmic pattern recognition receptor, and studies have found that most of its members can form a large molecular platform, such as NLRP1ASCASCCaspase-1 and caspase-5, a large complex that is defined as "inflammasome", the inflammatory body. It has been proved to be necessary for the activation of the inflammatory cytokine IL-1 尾-Interleukin-1 尾. After that, many receptor proteins of NLRs [2-5] family and PYHINs family have been proved to be able to form functional inflammatory corpuscles stimulated by various Damage-associated molecular patterns dabs and Pathogen-associated molecular patterns pampps. Now, the presentation of inflammatory bodies has been recognized as a landmark discovery of the intracellular immune surveillance system. Inflammatory corpuscles are widely involved in the innate immune response against microbial infection, among which the most widely studied is NLRP3(NLR family, pyrin domain containing 3) inflammatory corpuscles. Molecular targeting therapy based on NLRP3 inflammatory bodies is also being studied. Therefore, exploring the molecular regulation mechanism of NLRP3 signaling pathway may provide an important basis for revealing the relationship between inflammation, immunity and disease, and provide new targets for the treatment of some diseases. In recent years, the study of NLRP3 inflammatory bodies and their interacting proteins has increased the understanding of the regulation of NLRP3 function. Therefore, in order to further explore the signal regulation mechanism and biological function of inflammatory bodies, this study screened proteins interacting with NLRP3 by yeast two-hybrid technique. The interaction proteins of NLRP3 were screened from human leukocyte cDNA library using three basic domains of NLRP3, PYDX NACHTLRR as bait. After further verification in yeast cells, we screened 4 candidate positive clones interacting with NLRP3 from 87 primary positive clones, namely, ECHS1(enoyl CoA hydratase, short chain1, UBE2I(ubiquitinconjugating enzyme E2IXRPL34 ribosomal protein L34) and PXNPAPAXLIN. The interaction of these candidate proteins with NLRP3 was further confirmed in eukaryotic cells. Co-IPP immunoprecipitation assay showed that ECHS1 could bind to NLRP3. This study found that the interaction between NLRP3 and ECHS1 may be a new regulatory factor for NLRP3, and it is possible to find a new mechanism for the regulation of NLRP3 function by further study. 15 figures, 3 tables, 58 references
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3411

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