抑素蛋白(prohibitin)1调控蛋白酶激活受体(protease-activated receptor)1内化转运及降解的功能和机制

Protease-activated receptor-1 (PAR1) is the receptor of proteases such as thrombin and matrix metalloprotease 1 (MMP1) and is closely linked to inflammation, tumorigenesis and metastasis. Prohibitins (PHBs), being members of stomatin/prohibitin/flotillin/ HflK/C superfamily, are ubiquitously expressed and highly evolutionarily conserved proteins. There are two members of PHBs in mammals, namely PHB1 (32 kDa) and PHB2 (37 kDa). The absence of PHBs leads to severe phenotypes and deficiencies in higher eukaryotes, and PHB knockout is lethal in mice. Our recent innovative results show that PAR1 is the membrane receptor of amphibian trefoil factor (Bm-TFF2), a mucosal healing factor, and mediates its biological functions. Based on the clues, we further showed that protease-activated receptor 4 is able to mediate the mucosal healing function of human trefoil factor 2 (hTFF2) (Zhang Y et al., Cell Mol Life Sci 2011, 68, 3771-3780). Interestingly, along this line, our results revealed that down-regulation of PAR4 expression occurs frequently in human gastric cancers and exhibits association with more aggressive gastric cancer, suggesting the importance of putative TFF2-PAR4 signaling in mucosal healing (Zhang Y et al., Int J Biochem Cell Biol 2011, 43, 1277-1283). Surprisingly, using Bm-TFF2 as a molecular probe, for the first time, we found that PHBs are required in protease-activated receptor 1-mediated human platelet aggregation, which is different from mouse platelets. They were found to interact and co-localize with PAR1 in human platelet membrane and regulate PAR1-mediated platelet activation (Zhang Y et al., J Thromb. Haemosta 2012, 10，411-418). Our preliminary results on cultured cells further revealed that PHB1 is involved in the process of PAR1 internalization and degradation. Present project will focuses on: (1) exploring the unknown functions and biological significance of PHB1 in regulating PAR1 internalization, trafficking and degradation by using normal primary cultured cells and abnormal tumor cells; (2) clarifying the regulatory mechanisms of PHB1 in PAR1 trafficking and degradation, including the interaction between PHB1 and PAR1 and the contribution of each of PHB1 functional domains; (3) investigating the biological influence of PHB1 regulating PAR1 internalization, trafficking and degradation on the ability of oncogensis and metastasis of tumor cells in vitro and in vivo. The project could promote the understanding of the relationship between protease-activated PAR1 signaling pathway and PHBs, and the related molecular pathological significance in complex human diseases.

蛋白酶激活受体1 (PAR1)是凝血酶, 金属蛋白酶MMP1等蛋白酶的受体，与炎症及肿瘤的发生和转移密切相关。抑素蛋白（prohibitins， PHBs）高度保守。我们目前创新结果是: 阐明PAR1是两栖类三叶因子Bm-TFF2作用的膜受体。以Bm-TFF2为分子探针，首次发现PHBs与PAR1的联系及对PAR1受体激活的调节作用，初步揭示PHB1参与了PAR1的蛋白降解过程。本项目旨在：以正常原代培养的细胞和异常的肿瘤细胞为模型研究未知的PHB1调控 PAR1内化转运及降解的功能和生物学意义；揭示PHB1调节 PAR1转运及降解的作用机制，包括PHB1与PAR1的相互作用，PHB1各个功能结构域的贡献和作用；利用细胞和动物模型揭示PHB1调控 PAR1内化转运及降解对肿瘤细胞生长与转移的影响。促进深入解析蛋白酶激活的PAR1受体信号与PHBs蛋白的关系及在人类复杂疾病中的分子病理意义。

蛋白酶激活受体(Protease activated receptors, PARs)属于G蛋白偶联受体家族(G protein coupled receptor, GPCR)，主要有4个成员，分别是PAR1，PAR2，PAR3和PAR4。其中PAR1主要作为凝血酶, 金属蛋白酶MMP1等蛋白酶的受体，广泛参与了凝血、炎症、肿瘤发生发展等重要生理病理过程！抑素蛋白(Prohibitins，PHBs)属于SPFH (stomatin/prohibitin/flotillin/ HflK/C) 家族, 其主要分布于细胞质膜，线粒体和细胞核等，广泛参与了信号通路调节，细胞增殖，细胞凋亡及线粒体功能的调节。.实验室前期以云南产两栖动物大蹼铃蟾中鉴定到的具血小板激动活性的单链三叶因子Bm-TFF2为研究对象，阐明PAR1作为其受体介导了其活性发挥，后续研究发现血小板膜PHB1可与PAR1相互作用，初步说明PHBs具有参与和调节PAR1受体激活的功能；然而血小板毕竟是无核细胞，其生命周期只有一次，PAR1激活后的信号过程在其上面无法研究，如PAR的内化转运及降解等，因此在其它有核细胞上进一步研究PAR1的内化转运及降解过程十分必要！所以就开展了本项目研究，.本项目旨在：（1）研究PHB1参与和调节 PAR1内化转运及降解的作用机制；（2）揭示PHB1调控 PAR1内化转运及降解在肿瘤细胞生长与转移中的影响。促进深入解析蛋白酶激活的PAR1受体信号与PHB1的关系，以及它们所整合的信号通路在人类复杂疾病中的分子病理意义。.项目按计划执行，进展顺利。在有核细胞（正常细胞HUVEC和肿瘤细胞MDA-MB-231）上最终阐释了PHB1在终止PAR1信号通路 (即受体内化转运及降解)过程中发挥了重要作用。一方面，肿瘤细胞PHB1在细胞膜定位的缺失可引起持续的PAR1信号激活;另一方面，细胞内高表达的PHB1可抑制PAR1降解使得肿瘤细胞具有高度侵袭性。该研究揭示了肿瘤细胞持续生长和高浸润性的分子病理新机制，也提供了在肿瘤恶性诊断和治疗中以PHB1作为新标示分子和作用靶点的可能性。.目前已发表标注该基金资助SCI论文16篇，其中影响因子大于5的6篇，包括影响因子大于10的杂志PNAS 1篇；.“基于生物生存策略的有毒动物中药功能成分定向挖掘技术体系”获得2013年度国家技术发明二等奖。