MRP8内化的分子机制及其生物学功能研究

发布时间：2018-03-27 21:01

本文选题：人髓样相关蛋白8　切入点：脓毒症　出处：《南方医科大学》2010年硕士论文

【摘要】：人髓样相关蛋白8 (myeloid-related protein-8,MRP8)是钙结合蛋白S100家族成员之一,具有EF手型结构。MRP8相对分子质量比较小,其单体由2个EF手Ca2+结合区和与之相连的中央铰链区组成,形成螺旋-环-螺旋(Helix-Loop-Helix, H-L-H)结构。其中位于C端的EF手型基序与Ca2+亲和力较高,与Ca2+结合后,该蛋白的构象发生改变,暴露出与靶蛋白相互作用的位点,从而发挥相关的生物学功能。MRP8最早发现于中性白细胞中,被认为是一种具有免疫原性的蛋白。随着研究的深入,发现该蛋白与炎症高度相关,能够募集单核细胞至炎症灶处,是许多急性和慢性炎症的重要促炎因子。另外MRP8／MRP9形成异二聚体在炎症中起了重要作用,该蛋白的血清浓度与炎症活动有关,因此,临床上常用其作为监测疾病发展变化及治疗愈后的指标。脂多糖(lipopolysaccharide, LPS)在革兰氏阴性菌感染的炎症反应中起到了至关重要的作用。该细菌产物能够引起包括巨噬细胞在内的机体免疫系统产生特异性反应。LPS介导的失控性机制的爆发会导致脓毒症或者是全身炎症反应综合症,而后者是一个非传染性的疾病。尽管采取了大量抗菌治疗,但仍然是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LPS等激活的单核／巨噬细胞和内皮细胞会大量释放MRP8,同时坏死组织细胞崩解也释放出大量的MRP8。释放到细胞外的MRP8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,MRP8在炎症反应过程中起着重要的作用。尽管目前对于MRP8的生物学功能尚未完全阐明,但是研究发现MRP8蛋白在炎症及肿瘤中的高表达并具有细胞因子样的效应,提示该蛋白在许多与炎症及肿瘤发生发展过程中具有重要的作用。因而MRP8参与炎症、应激等反应的分子基础,特别是作用于细胞相关信号过程成为近年来研究的热点。并且发现,MRP8能与细胞表面的硫酸肝素蛋白多糖(heparan sulfate proteoglycan, HSPG)及羧化聚糖发生相互作用。更重要的是研究发现CD36分子、晚期糖基化终末产物受体(receptor for advanced glycation end products, RAGE)可能是MRP8的细胞表面受体,然而,阻断这些受体并不能完全抑制MRP8的胞内信号传递,这提示细胞表面可能还存在其他的MRP8受体或信号转导方式。结合近年来人们陆续发现一些细胞因子、生长因子或其受体能够被内化而参与胞内的信号反应,我们推测MRP8也能够被效应细胞内化,且其内化活性与其丰富的生物学功能密切相关。内吞是一个复杂并且高效的过程,细胞能够摄取营养物质并且与外界进行联系。内吞小泡的形成过程非常复杂,首先外源性的蛋白有序的富集于细胞膜周围,紧接着质膜上的脂质双分子层发生巨大的变形重构将蛋白包裹形成凹陷的吞噬小泡。内化的过程不仅能够从外界获得物质与能量,同时也能够调节细胞表面某些受体的数量,包括信号通路受体、粘附分子和GPI偶联受体。外源性物质进入细胞的方式主要有三种,经典的依赖包涵素的内吞途径(clathrin-dependent endocytosis),即生物大分子通过与AP-2和其他的受体相互作用而进入细胞。很多生物大分子,包括低密脂蛋白(low density lipoprotein,LDL)、转铁蛋白(transferrin)、表皮生长因子(epidermal growth factor, EGF)、胰岛素等都是通过受体介导的内吞作用内化的。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。胞膜窖是一种表面分布着特定受体,富含胆固醇及特征性小窝蛋白(caveolin)的微结构。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(cholera toxin, CTx)和猿病毒40 (simian virus 40, SV40)等的内化过程。另一种包涵素非依赖的途径,主要是一些与GPI受体家族结合的分子和糖脂类物质,这类物质具有多形的结构并且不具备坚固的外壳。硫酸肝素蛋白多糖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,本研究首先构建MRP8与增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)融合表达载体,利用共聚焦显微镜观察MRP8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MRP8绿色荧光蛋白共同孵育,研究MRP8通过何种内化通路进行内化;之后通过生物信息学的分析研究介导MRP8内化的功能域；进一步研究MRP8内化后的去路问题。最后利用LiquiChip-液相芯片技术检测MRP8经刺激后细胞因子的分泌情况,探讨MRP8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、MRP8以时间和能量依赖性方式先与胞膜结合,进而形成内吞小泡内化进入哺乳细胞。5min时,开始与细胞膜结合,1h左右达到平衡,9h基本代谢完全。第二、MRP8首先可能与细胞膜表面的HSPG上的HS链结合,并依赖其特异性受体,经胞膜窖介导的内吞途径进入细胞,而后细胞骨架参与了该蛋白的细胞内活动；第三、MRP8的内化必须依赖钙离子,由两个EF手型基序协同完成；第四、MRP8内吞进入细胞后,最终被泛素标记进入蛋白酶体降解。而该蛋白上第92位赖氨酸发生泛素化修饰是其降解的主要途径。第五、MRP8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,是机体对抗炎症的一项重要调节机制。 MRP8参与了全身炎症反应的过程,与许多炎症及肿瘤有关。本研究对MRP8的内化机制进行了初步研究,且证实MRP8可能通过内化降解,抑制其刺激细胞分泌炎症因子的功能。这些研究结果不但加深了我们对MRP8生物学功能的认识,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]:Human myeloid related protein 8 (myeloid-related protein-8 MRP8) is one of the members of the family of calcium binding protein S100, EF has the hand structure relative molecular mass of.MRP8 is relatively small, the monomer consists of 2 EF Ca2+ binding region and the central hinge connected with the area, the formation of helix loop - helix (Helix-Loop-Helix H-L-H) structure. Which is located in the C end of the EF hand motif Ca2+ with higher affinity, when combined with Ca2+, the protein conformation changes, exposed and target protein interaction sites, and thus play biological functions related to.MRP8 was first discovered in white blood cells, is considered as a kind of protein immunogenic. With further research, found the protein with inflammation is highly correlated to the recruitment of monocytes to inflammatory lesions, is an important proinflammatory factor of many acute and chronic inflammation. In MRP8 / MRP9 formation of two dimers in inflammation play The serum concentration of the protein is associated with inflammatory activity, so it is used clinically as a marker for monitoring the development and healing of the disease.
Lipopolysaccharide (lipopolysaccharide, LPS) on the inflammatory reaction of gram negative bacterial infection plays a very important role. The bacterial products can cause the body's immune system including macrophages, have lost control of the mechanism of specific reaction mediated by.LPS will lead to the outbreak of sepsis or systemic inflammatory response syndrome, which is a non infectious disease. Despite numerous antimicrobial treatment, but is still one of the major causes of death of critical patients, the mortality rate is as high as 30 to 70%. over the past ten years of clinical use of tumor necrosis factor (tumor necrosis, factor, TNF) and interleukin 1 (interleukin-1, IL-1) are not satisfactory antagonist treatment results. Recent studies show that inflammatory stimulation LPS activation of monocytes / macrophages and endothelial cells release a lot of MRP8 at the same time, necrotic tissue cell disintegration also release The release of MRP8 molecules of MRP8. released into the extracellular through further activation of monocytes or endothelial cells such as induced expression and release of inflammatory factors and adhesion molecules, including lead and participate in the brain, lung, gastrointestinal tract, joint inflammation, injury of heart and other organs and extensive systemic inflammatory response even death. Thus, MRP8 plays an important role in the inflammatory process.
Although the biological function of MRP8 has not been fully elucidated, but the study found that high expression of MRP8 protein in inflammation and tumor and has the effect of cytokine, which plays an important role in suggesting that the occurrence of the protein in many inflammatory and tumor development. Thus MRP8 is involved in inflammation, the molecular basis of stress reaction, especially the effect on the cell signaling process becomes a hotspot in recent years. And it is found that MRP8 with cell surface heparan sulfate proteoglycan (heparan sulfate, proteoglycan, HSPG) and carboxylation chitosan interaction. More important is the study found that CD36 molecules, advanced glycation end product receptor (receptor for advanced glycation end products, RAGE) is a cell surface receptor of MRP8, however, blocking these receptors can not completely inhibit MRP8 intracellular signaling, suggesting that cell surface There may be other MRP8 receptor or signal transduction. In recent years, people have found some combination of cytokines, growth factors or their receptors can be internalized in response to intracellular signaling, we hypothesize that MRP8 can also be the effect of cellular internalization, biological function and its internalization activity was closely related to the rich.
Endocytosis is a complex and efficient process, cells can intake of nutrients and contact with the outside world. The forming process is very complex endocytic vesicles, the exogenous protein enriched in the orderly around the cell membrane, followed by lipid bilayer membrane on the deformation of reconstruction of giant protein inclusion formation in depression the phagocytic vesicle internalization process. Not only can obtain the material and energy from the outside, but also can regulate the cell number of some surface receptors, including signaling receptors, adhesion molecules and GPI coupled receptors. There are mainly three kinds of exogenous substances into cells, the classical endocytic pathway dependent inclusion pigment (clathrin-dependent endocytosis), namely biological macromolecules with AP-2 and other receptor interactions into cells. Many biological molecules, including low density lipoprotein (low density, lipoprotein, LDL Transferrin (transferrin), epidermal growth factor (epidermal), growth factor, EGF), insulin is internalized by receptor-mediated endocytosis. The nonclassical endocytic pathway does not depend on the inclusion of one of the most important is the caveolar endocytosis (caveolae) mediated by the cell membrane. The cellar is a surface distribution of a specific receptor, rich in cholesterol and characteristic of caveolin-1 (caveolin) micro structure. The first study this structure plays an important role in the cell signal transduction process, discovered in recent years for its involvement in various bacteria and virus particles, such as cholera toxin (cholera toxin, CTx and simian virus 40 (Simian) virus 40, SV40) internalization process. Another clathrin dependent way, is the main molecular and glycolipids and substance GPI receptor family with the structure of this kind of material is pleomorphic and with no preparation solid protein heparan sulfate shell. Polysaccharide is a kind of glycoprotein exists widely in cell surface and basement membrane by heparin sulfate (heparan, sulfate, HS) and core protein covalently linked to activation of specific receptors as co receptor ligands in the regulation of many cells, mechanical support, adhesion, movement, proliferation, and plays an important role in the the differentiation and morphogenesis. In addition, the research also indicates that HSPG also plays an important role in the internalization process, may be an important molecular mechanism of the internalization of universal.
Based on the above understanding, this study constructed MRP8 and enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) fusion expression vector, using a confocal microscope to observe whether MRP8 has internalized activity; then use a series of related pathway inhibitors and internalization of fluorescent dyes and MRP8 green fluorescent protein were incubated on MRP8 by what kind of internalization pathway after internalization; through the bioinformatics analysis of MRP8 domain mediated internalization; further research on the way of MRP8 after internalizing problems. Finally using LiquiChip- liquid chip technology to detect MRP8 by secretion of cytokines after stimulation, investigate the internalization of MRP8 correlation with inflammation, lay the foundation for further study further.
Through the above research, we obtained the following conclusions. First, MRP8 binding and cell membrane in time and energy dependent manner, and the formation of endocytic vesicles internalized into mammalian cell.5min, began to combine with the cell membrane, 1H balance, 9h basic metabolism. Second, MRP8 may be the first surface and cell membrane the HSPG HS chain with, and depends on its specific receptor, enter the cell through the endocytic pathway of caveolae mediated, and cytoskeleton proteins involved in the intracellular activities; third, MRP8 internalization must rely on calcium, completed by two EF hand motif fourth, MRP8 collaboration; Swallow after entering the cell, eventually the ubiquitin tag into proteasomal degradation. The protein of ninety-second lysine ubiquitinated is the main pathway for its degradation. Fifth, internalization and proinflammatory effect of MRP8 is accomplished through two different signal pathways The purpose of internalization is to degrade and inhibit the continuous aggravation of inflammation, which is an important regulatory mechanism for the body against inflammation.
MRP8 is involved in the process of the systemic inflammatory response, many associated with inflammation and tumor. In this study, internalization mechanism of MRP8 was studied, and confirmed by MRP8 might inhibit the degradation of internalized function, stimulate cells to secrete inflammatory cytokines. These results not only deepen our understanding of the biological function of MRP8, but also may from the fundamental effects and changes in clinical sepsis and understanding and treatment of uncontrolled systemic inflammation, so it has very important theoretical basis and clinical significance.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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