MRP14内化的分子机制及其生物学功能研究

发布时间：2018-05-20 18:00

本文选题：人髓样相关蛋白14 + 脓毒症　；参考：《南方医科大学》2009年硕士论文

【摘要】：人髓样相关蛋白14(myeloid-related protein-14,MRP14)是钙结合蛋白S100家族成员之一。MRP14相对分子质量比较小,其单体由两个EF手型基序组成,分别位于氨基末端和羧基末端,此结构为钙离子结合位点。其中,位于羧基端的EF手型基序与钙离子结合力较强,与钙离子结合后,其蛋白构象发生改变,从而暴露出与靶蛋白的结合位点,通过与靶蛋白作用而发挥其生物学效应。MRP14具有丰富的生物学效应,如参与蛋白质的磷酸化,细胞的增殖与分化,细胞骨架构建,调节细胞内钙离子的稳态以及广泛参与全身的炎症反应和组织损伤等。脓毒症仍然是目前临床危重患者的重要死因之一,其死亡率高达30～70%。过去十几年中临床利用肿瘤坏死因子(tumor necrosis factor,TNF)和白细胞介素1(interleukin-1,IL-1)拮抗剂治疗都未取得满意效果。近年来的研究表明,致炎刺激脂多糖(lipopolysaccharide,LPS)等激活的单核/巨噬细胞和内皮细胞会大量释放MRP14,同时坏死组织细胞崩解也释放出大量的MRP14。释放到细胞外的MRP14分子通过进一步激活单核或内皮等细胞,引起大量炎症因子和黏附分子的表达和释放,导致并参与了包括脑组织、肺、胃肠道、关节、心脏等多种脏器的炎症损伤以及广泛的全身性炎症反应甚至死亡。由此可见,MRP14在炎症反应过程中起着重要的中枢作用。 MRP14参与炎症反应的分子基础及其相关信号过程近年来成为国内外的研究热点,也取得了一些进展,但很多过程仍不明确,特别是MRP14作用于细胞的信号机制。近年研究者们陆续鉴定出MRP14的一些细胞表面受体,包括晚期糖基化终末产物受体(receptor for advanced glycation end products,RAGE)、toll样受体(toll-like receptor,TLR)家族成员等,但阻断所有这些受体仍不能完全抑制MRP14对细胞的作用,表明细胞表面可能还存在其它受体或其它可能的信号转导方式。MRP14在炎性细胞有大量表达,结合近年来人们陆续发现一些细胞因子、生长因子或它们的受体可以被细胞内化(internalization)而参与后续信号反应,我们推测MRP14可能也可以被效应细胞内化,且其内化活性与其丰富的生物学功能密切相关。细胞和外界进行着广泛的物质交换。除一些小分子物质外,大多数生物大分子包括蛋白、多糖、多核苷酸以及一些病毒、细菌颗粒、坏死调亡细胞的产物都是通过内化的方式进入细胞,内化广泛参与人体各种生理和病理过程。内吞(endocytosis)是大分子物质内化的最重要的方式,是一个复杂的生物学过程,有多种信号通路参与,包含了广泛的蛋白与蛋白以及蛋白与脂质、糖类的相互作用。目前研究较清楚的特异性内化方式是经典的依赖包涵素的内吞途径(clathrin-dependent endocytosis),即生物大分子在相应受体介导下通过形成有包涵素(clathrin)包被的小泡而被内吞,很多生物大分子包括低密脂蛋白(lowdensity lipoprotein,LDL)、转铁蛋白(transferrin)、表皮生长因子(epidermalgrowth factor,EGF)、胰岛素等都是通过受体介导的内吞作用进行的。近年来研究较多的另一种重要的内吞途径是非经典的不依赖包涵素的内吞途径,其中最重要的是胞膜窖(caveolae)介导的内吞。胞膜窖是细胞膜上脂筏(lipid raft)的一种,是细胞膜上的一块瓶颈形内陷的膜结构,富含胆固醇、神经鞘磷脂以及特征性蛋白—窖蛋白1(caveolin-1)。最初研究这个结构在细胞信号转导过程中起重要作用,近年发现其参与了多种细菌和病毒颗粒,如霍乱毒素(choleratoxin,CTx)和猿病毒40(simian virus 40,SV40)等的内化过程。硫酸肝素蛋白多糖(heparan sulfate proteoglycan,HSPG)是广泛存在于细胞表面和基底膜的一类糖蛋白,由硫酸肝素(heparan sulfate,HS)和核心蛋白共价连接而成,它们作为共受体调节许多配体的特异性受体的激活,在细胞的机械支持、粘附、运动、增殖、分化和形态形成中起重要作用。除此之外,研究还表明HSPG在内化过程中也具有重要作用,可能是一种普遍的内化机制中的重要分子。基于以上认识,本研究首先将MRP14与增强型绿色荧光蛋白(enhancedgreen fluorescent protein,EGFP)融合表达,通过荧光定位观察方法检测MRP14是否具有内化活性;接着利用一系列与内化通路相关的抑制剂和荧光染料与带有绿色荧光的MRP14蛋白共同孵育细胞,通过荧光共定位方法阐明MRP14通过何种内化通路进行内化;之后在生物信息学基础上通过构建MRP14上不同结构域片断与EGFP的融合表达载体,利用荧光显微镜研究介导MRP14内化的功能域;进一步我们运用类似方法对MRP14内化后的去路问题进行了研究;最后,我们利用liquichip-液相芯片技术检测MRP14刺激后细胞因子的产生情况,从而初步探讨MRP14内化与炎症的相关性,为后续进一步的深入研究打下基础。通过以上研究,我们得到了如下结论。第一、MRP14以时间和能量依赖性方式通过内吞作用被哺乳动物细胞内化,内吞过程在15 min时已经开始,1 h左右达到平衡,内吞的MRP14颗粒散在分布于细胞质内;第二、MRP14可能通过与细胞膜表面HSPG的HS链相互作用,并在其特异性受体协助下,通过依赖胞膜窖的内吞途径进入细胞,之后内吞小泡在细胞骨架协助下在细胞内运动,融合蛋白最终经泛素标记后,进入蛋白酶体降解;第三、MRP14的内化必须在钙离子存在的情况下,由两个EF手型基序协同完成;第四、MRP14的内化与促炎效应是通过两条不同的信号通路完成的,内化的目的是为了降解,从而抑制炎症的持续加重,是机体对炎症反应的一项重要调节机制。本研究对MRP14的内化机制进行了初步研究,且证实MRP14可能通过内化降解,抑制其刺激细胞释放炎症因子的功能。这些研究结果不但加深了我们对MRP14生物学性质的认识,而且还有可能从根本上影响和改变临床上对脓毒症和失控性全身炎症的认识和治疗方案,因而具有极为重要的基础理论和临床治疗意义。
[Abstract]:The human myeloid related protein 14 (myeloid-related protein-14, MRP14) is one of the members of the calcium binding protein S100 family.MRP14 with relatively small relative molecular weight. The monomer consists of two EF hand types, which are located at the end of the amino group and the end of the carboxyl group. This structure is a calcium binding site. Among them, the EF hand motif at the carboxyl terminal and the calcium ion junction are located at the carboxyl terminus. After combining with calcium ion, the conformation of protein is changed and the binding site of the target protein is exposed. The biological effect of.MRP14 has a rich biological effect, such as the phosphorylation of protein, the proliferation and differentiation of cells, the construction of cytoskeleton, and the stability of intracellular calcium ion. State and extensive participation in systemic inflammatory response and tissue damage.
Sepsis is still one of the most important causes of death in critically ill patients at present. The mortality rate is up to 30 to 70%., and the clinical use of tumor necrosis factor (TNF) and interleukin 1 (interleukin-1, IL-1) antagonists in the past decade has not achieved satisfactory results. Recent studies have shown that inflammation stimulates lipopolysaccharide (lipopol). Ysaccharide, LPS) and other activated monocytes and macrophages and endothelial cells release a large number of MRP14, while necrotic tissue cells disintegrate and release a large number of MRP14. released to the extracellular MRP14 molecules by further activating mononuclear or endothelial cells, causing the expression and release of a large number of inflammatory factors and adhesion molecules, leading to and participating in the package. It can be seen that MRP14 plays an important central role in the process of inflammation, including the inflammatory damage of various organs such as brain tissue, lung, gastrointestinal tract, joint and heart, as well as extensive systemic inflammation and even death.
MRP14 participates in the molecular basis of the inflammatory response and its related signal process in recent years has become a hot spot at home and abroad, and some progress has been made, but many processes are still unclear, especially the signal mechanism of MRP14 on cell. In recent years, researchers have identified some cell surface receptors of MRP14, including late glycosylation end products. Receptor (receptor for advanced glycation end products, RAGE), toll like receptor (Toll-like receptor, TLR) family members, etc., but blocking all these receptors still can not completely inhibit the action of MRP14 to cells, suggesting that the cell surface may still have other receptors or other possible signal transduction pathways that have numerous tables in inflammatory cells. In recent years, a number of cytokines have been discovered, and growth factors or their receptors can be involved in cell internalization (internalization) to participate in subsequent signal reactions. We speculate that MRP14 may also be internalized in effector cells, and its internalization activity is closely related to its rich biological function.
In addition to some small molecular substances, most biological macromolecules include proteins, polysaccharides, polynucleotides, and some viruses, bacterial particles, and necrotic cells that enter cells through internalization, and are extensively involved in various physiological and pathological processes in the human body. Endocytosis (endocytosis ) is the most important way of macromolecular substance internalization. It is a complex biological process with a variety of signaling pathways involved, including a wide range of proteins and proteins, and the interaction of proteins with lipids and carbohydrates. The most clear specific internalization is the classic inclusion element endocytic pathway (clathrin-dependent endocy). Tosis), that is, biologic macromolecules are endocytosis by the corresponding receptor mediated vesicles that form inclusion (clathrin) inclusion. Many biological macromolecules include low density lipoprotein (lowdensity lipoprotein, LDL), transferrin (transferrin), epidermal growth factor (epidermalgrowth factor, EGF), insulin, etc. The other important endocytic pathway, which has been studied more recently, is an endocytic endocytic approach that is not dependent on inclusion elements. The most important is the endocytic endocytosis mediated by caveolae. The cell membrane cellar is a kind of lipid rafts (lipid raft) on the cell membrane. It is a membrane structure of a bottleneck on the cell membrane, which is rich in cholesterol. Nerve sphingomyelin and characteristic protein - caveolin 1 (caveolin-1). The initial study of this structure plays an important role in cell signal transduction. In recent years, it has been found to be involved in the internalization of a variety of bacteria and virus particles, such as cholera toxin (choleratoxin, CTx) and ape virus 40 (simian virus 40, SV40). Heparin sulfate polysaccharide (he) Paran sulfate proteoglycan, HSPG) is a kind of glycoprotein that widely exists on the surface of the cell and the basement membrane, which is covalently linked by heparin sulfate (heparan sulfate, HS) and core proteins. They are activated by the specific receptors regulating many ligands by common receptors, in cell mechanical support, adhesion, exercise, proliferation, differentiation and morphogenesis. In addition, the study also shows that HSPG plays an important role in internalization, and may be an important molecule in the internalization mechanism.
Based on the above understanding, this study first fused MRP14 with enhanced green fluorescent protein (enhancedgreen fluorescent protein, EGFP), and detected whether MRP14 had internalization activity by fluorescence localization observation, and then used a series of inhibitors and fluorescent dyes associated with internalized pathways and the MRP14 protein with green fluorescence. With the incubating cells, the internalization of MRP14 through the fluorescence co localization method is clarified, and then on the basis of bioinformatics, the fusion expression vector of different domain fragments on MRP14 and EGFP is constructed, and the functional domain of MRP14 internalization is studied by fluorescence microscopy. Further we use similar methods to internalize MRP14. The following problem is studied. Finally, we use liquichip- liquid phase chip technology to detect the production of cytokines after MRP14 stimulation, so as to preliminarily discuss the correlation between MRP14 internalization and inflammation, and lay a foundation for further further in-depth study.
Through the above study, we get the following conclusions. First, MRP14 is internalized in mammalian cells by endocytosis in a time and energy dependent manner. Endocytosis begins at 15 min and balances at about 1 h. The endocytic MRP14 particles are scattered in the cytoplasm; second, MRP14 may pass the HS chain to the cell membrane surface HSPG. Interaction, assisted by its specific receptor, enters cells by endocytosis pathway dependent on the cell membrane cellar, and then the endocytic vesicles move within the cell with the help of the cytoskeleton. The fusion protein is eventually labeled by ubiquitin and enters proteasome degradation; third, the internalization of MRP14 must be based on the presence of calcium ions by two EF hand groups. Fourth, the internalization and proinflammatory effect of MRP14 is accomplished through two different signaling pathways. The purpose of internalization is to degrade and inhibit the continuous aggravation of inflammation, which is an important regulatory mechanism for the body's inflammatory response.
This study has conducted a preliminary study on the internalization mechanism of MRP14, and confirmed that MRP14 may inhibit the release of inflammatory factors by its endogenous degradation. These results not only deepen our understanding of the biological properties of MRP14, but also have a fundamental effect on the clinical response to sepsis and out of control all over the body. The understanding and treatment of inflammation is of great importance in basic theory and clinical treatment.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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