利用亲和磷酸蛋白组学筛选和鉴定氧化应激重塑型LPS信号通路中的新信号分子

发布时间：2018-01-12 01:35

本文关键词：利用亲和磷酸蛋白组学筛选和鉴定氧化应激重塑型LPS信号通路中的新信号分子　出处：《第一军医大学》2006年博士论文　论文类型：学位论文

【摘要】： 前期研究发现，缺血／再灌注损伤产生的氧化应激可以引发机体对第二刺激物(通常为细菌脂多糖)产生强烈得多的反应，从而使得病人更容易发生多器官功能衰竭综合征，此即为所谓的“两次打击”理论。迄今为止，尽管有相当多的研究针对氧化应激诱导的信号通路或经典的LPS信号通路在感染中的作用作了相应探讨，然而，这些研究很少涉及氧化应激和LPS信号通路的相互增效效应。实际上，在预先存在氧化应激的状态下，细胞内的LPS信号通路和传统意义上的LPS信号通路有很大区别。体内和体外研究均证实，氧化应激状态可以引发巨噬细胞针对LPS刺激分泌更多的细胞因子和趋化因子，从而导致过度的免疫反应。此外，，不同于传统LPS信号通路，这种激活作用应归因于src／PI-3K介导的超量NF-kappa B核转位。据此，先前的文献提出了“氧化应激可以导致细胞内LPS信号通路发生重塑”这一理论，然而，由于对参与这种重塑型LPS信号通路的信号分子知之甚少，我们难以准确地阐明其机制。迄今为止，还没有文献报道系统地比较传统LPS信号通路和氧化应激下发生重组的LPS信号通路的差别。为了更加全面和系统地研究氧化应激状态下的LPS信号通路，本文以PMA诱导分化的人外周血Thp-1巨噬细胞为模型，比较LPS刺激的细胞在有或无氧化应激提前处理的情况下磷酸化蛋白质组的差异，力图筛选更多潜在的信号分子。通过BD公司的PMAC磷酸化蛋白纯化柱，本研究富集了高达400μg磷酸化蛋白，与此同时，改良的分级染色法大大提高了检测灵敏度。经过这些方法的优化，我们最终在双向电泳图谱上检测到了大约1150个磷酸化蛋白斑点，远远超过此前报道的约700个，增加了低丰度的调节分子被检出的可能性。相对于仅用LPS刺激的Thp-1细胞，在有氧化应激提前刺激的Thp-1细胞中，有29个磷酸化蛋白斑点在双向电泳图谱中表现出重复的差异，其中，16个斑点发生了上调(包括新出现的斑点)，11个发生了下调(包括消失的斑点)，另外的2个在双向电泳图谱中的位置坐标发生了变化。我们选择了这些差异蛋白进行了质谱分析，到目前为止鉴定出了其中5个，这些蛋白参与多种多样的细胞过程例如蛋白质降解、信号转导和蛋白质折叠等。其中，proteasome beta-4亚基在有氧化应激时下调明显，而核基质蛋白NMP-238明显上调。此前曾有报道表明蛋白酶体在不同水平上参与传统LPS信号通路的调节：1，通过泛素-蛋白酶体通路负调节p38的活性；2，通过此通路负调节IRAK的活性；3，与LPS分子的直接结合，并通过糜蛋白酶活性将细菌抗原呈递给MHC 1类分子。而核基质蛋白NMP-238是激活Wnt／beta-catenin调控基因转录的一个关键辅助因子，而最近的研究也显示Wnt／beta-catenin介导一些感染过程。因此，结合前期的研究，我们提出以下假说，除src／PI-3K／NF-kappaB外，蛋白酶体和核基质蛋白NMP-238也很有可能参与氧化应激下LPS通路的重塑，主要体现在下列水平上：1，蛋白酶体活性急剧降低，从而导致p38活性强烈升高，刺激产生更多的黏附分子和细胞因子；2，急剧下降的蛋白酶体活性解除了对IRAK的负调控，从而引起无限制的IRAK激活，导致下游的JNK／AP-1和NF-kappaB通路广泛激活；3，下调的蛋白酶体活性减少了细菌的抗原呈递，从而降低了机体对细菌感染的获得性免疫能力。4，上调的NMP-238通过Wnt／beta-catenin通路加剧了氧化应激下LPS通路的炎症反应。综上所述，以上多种因素协同作用，从而激发机体在氧化应激条件下产生对LPS异常强烈的免疫反应，并导致机体对细菌LPS的易感性大大增加。目前，我们已开始对蛋白酶体和核基质蛋白NMP-238在氧化应激性LPS信号通路中的作用进行深入的功能研究，以验证上述假说是否成立。与此同时，我们正采取分泌组学和膜蛋白组学的策略以筛选更多的调节分子并随之开展进一步研究。
[Abstract]:The preliminary study found that oxidative stress, ischemia / reperfusion injury can lead to the body of the second generation of stimuli (usually lipopolysaccharide) an exaggerated response, which makes the patients more prone to multiple organ failure syndrome, the so-called "two hit" theory. So far, despite the role LPS signal pathway of considerable research on the classical signal pathway or induced by oxidative stress in infection in the study, however, the mutual synergistic effect of these studies rarely involve oxidative stress and LPS pathway. In fact, in advance of the oxidative stress state, intracellular LPS signal pathway of LPS signal pathway and the traditional sense is very different. In vivo and in vitro studies have confirmed that oxidative stress can induce macrophage in LPS stimulated the secretion of cytokines and chemokines more In addition, it is different from the traditional LPS signaling pathway, which should be attributed to the Src / PI-3K mediated overdose NF-kappa B nuclear transposition.
Accordingly, previous literature has proposed the theory that oxidative stress can lead to remodeling of intracellular LPS signaling pathway. However, because of little knowledge of signal molecules involved in this remodeling LPS signaling pathway, it is difficult for us to elucidate its mechanism accurately.
So far, no LPS signal pathway of recombination between traditional LPS signal pathway and oxidative stress are reported in the literature systematically under the difference. In order to more comprehensive and systematic study of LPS signaling pathway under oxidative stress, the PMA induced differentiation of human peripheral blood Thp-1 macrophage cells as a model, LPS stimulation the cells in the presence or absence of oxidative stress in advance when processing differences of phosphoproteome, to screening more potential signaling molecules. Purified through phosphorylation of PMAC protein of BD, this study enriched up to 400 protein phosphorylation of G, at the same time, the improved graded staining method and improve detection sensitivity. Through the optimization of these methods, we finally in electrophoresis detected about 1150 phosphorylated protein spots, far more than previously reported about 700, increased regulation of low abundance The possibility of a molecule being detected.
Compared with only using LPS stimulated Thp-1 cells, oxidative stress in LPS stimulated Thp-1 cells, 29 phosphorylated protein spots showed repeated differences in electrophoresis which occurred by 16 spots (including new spots), 11 embryos (including the disappearance of the spots 2), changes in the electrophoresis in position coordinates. Also we chose these proteins of mass spectrometry, so far identified 5 of them, these proteins involved in various cellular processes such as protein degradation, signal transduction and protein folding. Among them, proteasome beta-4 the subunit was significantly down regulated in oxidative stress, and nuclear matrix protein NMP-238 was up-regulated. Earlier reports suggest that regulation of proteasome is involved in the traditional LPS signal pathway on different levels: 1, through the ubiquitin protein Enzyme pathway negatively regulates p38 activity; 2, through this pathway negatively regulates the activity of IRAK; 3, direct binding of LPS molecules, and bacterial antigens to MHC 1 molecules by chymotrypsin activity. The nuclear matrix protein NMP-238 is a key auxiliary factor activating Wnt / beta-catenin regulation of gene transcription. Recent studies also show that Wnt / beta-catenin mediated some infection process. Therefore, based on the previous research, we propose the following hypothesis, in addition to the Src / PI-3K / NF-kappaB, the proteasome and nuclear matrix protein NMP-238 is likely involved in oxidative stress remodeling under the LPS pathway, is mainly reflected in the following levels: 1, proteasome activity decreases sharply, resulting in strong p38 activity increased, stimulates the production of adhesion molecules and cytokines more; 2, the proteasome activity decreased sharply to relieve the negative regulation of IRAK, resulting in no The limitation of IRAK activation, resulting in JNK / AP-1 and NF-kappaB pathways downstream of extensive activation; 3, proteasome reduces bacterial antigen presentation, thereby reducing the body of bacterial infection acquired immunity.4, upregulation of NMP-238 through Wnt / beta-catenin pathway exacerbated inflammatory response of LPS pathway under oxidative stress. In summary, these factors synergy, so as to stimulate the immune response strongly on LPS under oxidative stress, and lead to susceptibility to bacterial LPS greatly increased. At present, we are beginning to function to further study the effect of proteasome and nuclear matrix protein NMP-238 in LPS signaling pathway in oxidative stress and to test this hypothesis is established. At the same time, we are taking secretome and membrane proteomics strategy to regulate molecular screening more and more Further research is carried out.

【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R363

【参考文献】