| 其他摘要 | 摘要 抑素蛋白（Prohibitin，PHB）是一类泛素化、功能众多并广泛分布的蛋白，PHB在细胞中的分布主要是在线粒体、细胞核及细胞膜的脂筏中。PHB参与了许多重要生理病理功能，如维持线粒体的稳定性，调控细胞周期与凋亡等，膜上的PHB还作为受体参与了与其它外界分子的识别结合及信号转导过程。肿瘤细胞侵袭和转移是恶性肿瘤的主要特征，与肿瘤的恶性水平及肿瘤患者的预后有着很大关系。PHB与肿瘤之间的关系已有广泛研究，然而却一直存在争议。蛋白酶激活受体（Protease activated receptor，PAR），是一类可被多种蛋白酶水解激活的膜受体，属于G蛋白偶联受体家族（GPCR）。在肿瘤微环境中，广泛分布有众多蛋白酶（如凝血酶等），这些蛋白酶可激活肿瘤细胞上的PARs，从而介导着肿瘤细胞的迁移浸润过程。目前研究表明PAR1信号通路激活紊乱与慢性炎症以及肿瘤发生发展及肿瘤浸润转移密切相关，其在肿瘤细胞的高表达和持续激活是导致肿瘤生长和转移的重要原因之一。然而目前关于PHB或PAR之间关系研究少之甚少，两者在肿瘤浸润性调节过程中的协调关系还从未研究过。目前的研究中，我们发现在恶性程度较高的乳腺癌细胞MDA-MB-231中，总PHB的表达是明显上调的，而其膜上PHB的表达却是缺失的；然而正常体细胞（HUVEC）却并没有发现这种奇怪的表达模式。提示膜上PHB可能参与了肿瘤细胞的某些标志性特征，如浸润能力等。后续对细胞膜PHB及胞内PHB的相关功能进行了解析，发现膜PHB主要以分子伴侣角色发挥功能，调节了PAR1的激动型内化转运过程；而胞内PHB主要是保护内化转运入胞的PAR1不被溶酶体降解掉。MDA-MB-231细胞因其膜PHB缺失使得激活后的PAR1无法进行激动型内化转运；胞内高表达的PHB又使得内化转运进来的PAR1不能进入溶酶体途径进行降解，最终导致了PAR1信号的持续激活；后续通过迁移和侵袭实验证实了正是因为这种信号的持续活化才导致了MDA-MB-231细胞具有更高的浸润性。细胞膜对于界定细胞的边界并维持所有活细胞的形态及功能是必需的。正因如此，细胞膜很容易受到攻击以至于在进化过程中使得它们逐渐成为一个倍受青睐的攻击靶点。孔道形成蛋白（Pore-forming proteins, PFPs）就是这样一类可以攻击膜的分子，它们可在宿主细胞膜上形成孔道而造成靶细胞的损伤和死亡。产气单胞菌溶素（Aerolysin）家族是β-PFPs中仅次于CDCs家族的第二大家族，其主要代表是由致病菌嗜水气单胞菌产生的孔道形成毒素——产气单胞菌溶素aerolysin。随着大规模基因组测序及生物信息学分析的广泛应用，在除细菌外的生物界中也发现了aerolysin类似蛋白（aerolysin-like proteins，ALPs）的存在，并认为ALPs存在于所有的生物体中。本实验室长期以云南两栖动物大蹼铃蟾（Bombina maxima）皮肤分泌物中分离得到的一个BmALP1与三叶因子（BmTFF3）复合物βγ-CAT为研究对象，近期工作发现βγ-CAT可以激活炎症小体而调动天然免疫，最终保护宿主抵御微生物感染，然而其发挥功能的受体及作用机制还不清楚。脂筏作为细胞膜上最重要的功能区域，参与了受体识别结合及后续的信号过程。脂筏中除了含有一些重要的蛋白（包括细胞膜上的绝大多数受体、通道等）外，细胞膜的糖鞘脂也是主要分布在脂筏中，并且同许多脂筏上的蛋白一样，扮演着受体分子的角色。本研究中我们就发现：细胞膜脂筏中的酸性糖鞘脂可能作为βγ-CAT的作用受体。首先我们发现唾液酸酶处理红细胞会抑制βγ-CAT引起的溶血，此外加入游离的唾液酸可完全抑制βγ-CAT的溶血，提示细胞膜表面唾液酸分子可能参与了βγ-CAT的功能；随后经过鉴定发现细胞膜上含唾液酸的酸性糖鞘脂（主要是神经节苷脂）才是βγ-CAT真正相互作用的分子，此外，结合脂类ELISA和体外分子相互作用的检测结果，发现βγ-CAT与不同类型的神经节苷脂具有直接结合能力，进一步研究表明βγ-CAT是与神经节苷脂的酸性糖链部分结合；此外，借助于体外生物分子相互作用检测技术，初步发现βγ-CAT的TFF结构域可能参与了与神经节苷脂的结合。其次，为了证实酸性糖鞘脂是不是βγ-CAT的作用受体，我们做了抗体阻断实验和神经节苷脂敲除实验。GM3类型的神经节苷脂是非神经组织中最主要的一类神经节苷脂，而且干扰掉GM3合成酶不仅会直接影响到GM3的合成，更会间接地影响所有类型神经节苷脂的合成。因此，本实验中我们选择以GM3作为对象分子。结果发现：GM3抗体与人红细胞孵育后发现βγ-CAT引起的红细胞溶血减弱，通过流式细胞观察发现结合到红细胞膜上的βγ-CAT的比例也有显著性降低；使用GM3合成酶的shRNA慢病毒颗粒感染THP-1细胞以沉默掉GM3的表达后发现，βγ-CAT引起的THP-1细胞的IL-1β分泌减少，且βγ-CAT对THP-1细胞的细胞毒性变弱，这些细胞学证据表明细胞膜上的酸性糖鞘脂特别是神经节苷脂很可能作为βγ-CAT发挥功能的直接受体或辅助受体。综上所述，我们首次将抑素蛋白（PHB）和蛋白酶激活受体1（PAR1）两者联系起来来探讨它们之间的相互调节关系对于肿瘤浸润能力的作用，这使得PHB有望成为一个判断肿瘤恶性程度的标志分子并有可能应用在肿瘤的分子诊断方面；成功的鉴定出βγ-CAT作用受体是细胞膜上的酸性糖鞘脂（主要是神经节苷脂），为以后研究其它物种来源的aerolysin类似蛋白的相关生物学功能及受体奠定了基础。; Abstract Prohibitins (PHBs) are ubiquitous, evolutionarily conserved, pleiotropic proteins that are mainly localized in the lipid rafts of mitochondria, nucleus, and plasma membrane. PHB is involved in multiple physiological and pathological functions, for example, PHB have a role in the maintenance of mitochondrial function and protection against senescence, and as a regulator of cell-cycle progression and in apoptosis, etc. In addition, the membrane PHB is usually as receptor of some invasive pathogen participated in the process of recognition and signal transduction. The invasion and metastasis of tumor are the principal feature of cancer and have a very close relation with the prognosis of patients with tumor. There have been widely studied about the relation between tumor and PHB, but has been controversial.Protease activated receptors (PARs) are a family of seven transmembrane G protein-coupled receptors (GPCRs) that are unique in their activation because the cleavage of the N-terminal domain by a protease unmasks a sequence that acts as a tethered-ligand, binding intramolecularly to activate the receptor and initiate multiple signaling cascades. The PARs could be activated by proteases which are widely distributed in tumor microenvironment and been known as involved in the invasion and metastasis of tumor. It has been found that the abnormal signal activation of PAR1 have a tightly association with chronic inflammation, the development、invasion and metastasis of cancer. The high expression level and sustained activation is one of most important reasons leading to tumor progression and metastasis. However, there is only a very few study about the relation between PHBs and PAR1 and the role of PHBs and PAR1 in tumor invasion and metastasis was unknown so far. In the present study, our findings shown that a total PHB level was significantly up-regulated in the highly invasive MDA-MB-231 breast cancer cells, while the membrane PHB of MDA-MB-231 cells was absent; however, the weird expression patterns was not be found in normal somatic cells HUVECs. These findings prompt that the membrane PHBs maybe involved in some symbolic characteristics of tumor cells, such as invasion and metastasis. In the subsequent study, we found the membrane PHBs mainly as molecular chaperones involved in the PAR1 activated internalization, while the intracellular PHBs negatively regulated PAR1 degradation, the PAR1 degradation can be significantly inhibited by the high intracellular PHBs level. The absence of membrane PHB of MDA-MB-231 cells may have resulted in the lost of PHB-dependent regulation of PAR1 activated internalization and Erk1/2 phosphorylation persisted activated. Moreover, the PAR1 degration was significantly inhibited by the high intracellular PHBs of MDA-MB-231 cells, these events eventually led to the persisted PAR1 signal activation and highly invasive of MDA-MB-231 cells. At last, we proved that the sustained activated PAR1 signal was a predominant factor leading to a more higher invasive of MDA-MB-231 cells via cell migration and invasion assays.The plasma membrane defines the boundary of every living cell, and its integrity is essential for life. For this reason, the plasma membrane emerges as an ideal target for attack from others cells. Not surprisingly, many organisms have developed pore-forming molecules designed to disturb membrane integrity for a variety of purposes. Pore-forming proteins (PFPs) can induce damage and death of target cells by punching holes in their target cell membrane to alter their permeability. The aerolysin family are the second β-PFPs family only to CDCs family. The representative molecule of aerolysin family is aerolysin. Aerolysin is produced by Aeromonas species, but aerolysin-like proteins (ALPs) are present in both Gram-positive and Gram-negative bacteria, plants and eukaryotes. Bioinformatics analysis has revealed that the aerolysin-like proteins (ALPs) can be found in all kingdoms of life. In our previous study, an ALP named βγ-CAT, consists of a βγ-crystallin fused ALP namely B. maxima aerolysin-like protein 1 (BmALP1) and a three domain trefoil factor (BmTFF3) was isolated from the frog B. maxima skin secretions. The recent work shown that βγ-CAT can protect host against microbial infection by activating inflammasome and trigger innate immunity, however, the receptor and molecular mechanisms of βγ-CAT was unknown.Lipid rafts are the most important microdomains of plasma membrane and widely involved in the receptor recognition and signal transduction. There are many important proteins (receptors, channals) and glycosphingolipids located in the lipid rafts. In our present study, we found the acidic glycosphingolipids of cell surface act as the potential receptor of βγ-CAT. Firstly, we found βγ-CAT induced RBC hemolysis was largely attenuated by eliminating sialic acid residues in cell membranes with sialidases. In addition, βγ-CAT induced hemolysis was completely inhibited by free sialic acids. These findings prompt that sialic acid residues of cell surface involved in the functions of βγ-CAT. In the subsequently study, we found that acidic glycosphingolipids containing sialic acid, also named gangliosides, was the molecule βγ-CAT interact with. In addition, the direct interaction between βγ-CAT and different types of gangliosides can be observed by using lipid ELISA assay and fortebio octet red 96 systems. Further study shown that the acidic glycan of gangliosides was the binding site of βγ-CAT. And preliminary assays revealed that the TFF domain of βγ-CAT maybe involved in the binding with gangliosides by using fortebio octet red 96 systems.Secondly, the antibody block assay and ganglioside knock-down assays was executed for validate whether the ganglioside was the receptor of βγ-CAT or not. The GM3 type ganglioside is the primary ganglioside at the non-nervous tissue, and knock-down of GM3 Synthase not only down-regulated the expression level of GM3, but also silencing the expression of all other types of gangliosides, so GM3 was chosen as subject. An attenuated hemolysis of βγ-CAT can be observed after erythrocyte incubate with GM3 antibody, and the ratio βγ-CAT binding to the erythrocyte was significantly decreased by using flow cytometry. The IL-1β release of THP-1 cells induced by βγ-CAT was significantly decreased after silencing the expression of GM3 with GM3 Synthase shRNA (h) Lentiviral Particles. Moreover, the cytotoxicity of βγ-CAT was also significantly decreased after silencing the expression of GM3 with GM3 Synthase shRNA (h) Lentiviral Particles. All these findings shown that the acidic glycosphingolipids of cell surface maybe the receptor or co-receptor of βγ-CAT.In conclusion, our work elucidate the role of PHB with PAR1 in the tumor invasion and metastasis for the first time, and these findings may then serve as the foundation for future uses of PHB as a promising target for developing more effective cancer treatments or even in refining cancer diagnostics and prognoses. In addition, we successful mining the acidic glycosphingolipids (especially gangliosides) of cell surface maybe the receptor or co-receptor of βγ-CAT, and these findings provide clues and scientific basis for biological functions research and receptor exploration of ALPs from other organisms |
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