抑素蛋白对蛋白酶激活受体１的分子调控机制研究
文献类型：学位论文
| 作者 | 王严戒 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-11 |
| 授予单位 | 中国科学院大学 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 抑素蛋白 凝血酶 蛋白酶激活受体 1 信号传导 拮抗剂 内化 降解 |
| 其他题名 | Mechanisms of prohibitins in regulating protease activated receptor 1 |
| 学位专业 | 动物学 |
| 中文摘要 | 抑素蛋白（prohibitins, PHBs）有两个同源家族成员，即抑素蛋白1 (PHB1)与抑素蛋白2 (PHB2)，它们表达广泛并且相对比较保守，从酵母到人都有它们的表达。曾有报道称细胞膜上的PHBs参与了伤寒、肥胖及肿瘤转移等。蛋白质组学发现血小板中存在PHBs, 但是对于它们在血小板中发挥的功能目前还不是很清楚。在我们目前的研究中，通过流式检测及激光共聚焦检测，发现PHB1及PHB2在血小板膜上有表达。通过蔗糖密度梯度离心，我们发现PHB1/2存在于血小板膜上的脂筏组分中。并且通过Bm-TFF2（一种PAR1的激动肽）的亲和层析分析、免疫共沉淀及激光共聚焦，我们发现PHBs可以与蛋白酶激活受体1（protease activated receptor 1, PAR1）结合及共表达。使用抗PHB1及抗PHB2的特异抗体或抗体的Fab段分别阻断PHB1及PHB2后，低剂量凝血酶（0.05U/ml）及PAR1激动肽（PAR1-AP, 20?M）引起的血小板聚集、?IIb?3的活化、颗粒释放及钙释放等血小板的活化过程均被抑制甚至消除，而对高剂量凝血酶（0.6U/ml）及PAR4激动肽（PAR4-AP, 300?M）引起的这些血小板活化过程没有影响。通过专一针对PHB1及PHB2的siRNA对细胞进行RNA干扰降低PHBs的表达后，MEG-01中的由PAR1-AP引起的钙释放显著降低。因此，我们认为PHBs是血小板上PAR1信号通路的新的未知的调节因子， PHBs可能会成为新的抗血小板治疗的有效靶点。 PHB1是一类具有多种功能并且与多种疾病相关的蛋白，然而目前还没任何一种针对PHB1的抑制剂或拮抗剂产生，从而导致对PHB1的研究进展缓慢。在这个基础上，我们从一个五个氨基酸组成的五肽库（205 =三百二十万个）中筛选出了一种可以与PHB1结合的小肽分子Pep5。Pep5的模式与其他与PHB1结合的蛋白所具有的模式类似，而且通过Pep5的亲和层析及Pep5与PHB1的分子对接，我们发现Pep5可以与PHB1结合。同时，Pep5能够专一的抑制PAR1-AP或低剂量凝血酶引起的血小板聚集或钙释放，IC50=200?????而对PAR4-AP或collagen引起的血小板聚集没有影响。因此，通过我们目前的研究，我们认为PHB1可以作为抗血小板治疗的有效靶点，而且通过进一步的序列优化及分子修饰，Pep可以成为PHB1的有效的拮抗剂。 正如我们前一部分所讲, PHB1参与了PAR1介导的血小板聚集，并且在血小板及MEG-01中对于PAR1相关信号通路的活化是必需的。然而，对于PHB1是否参与了PAR1其他的生物学过程，如去敏、内化转运、降解等PAR1信号终止等相关过程，目前还不是很了解。我们发现，在血管内皮细胞HUVEC及乳腺癌细胞MDA-MB-231中，通过RNA干扰去掉PHB1后，并不影响PAR1介导的钙释放，说明在这两类细胞中，PHB1并未参与调节PAR1介导的信号通路活化，在这两类细胞中PHB1对于PAR1信号通路的活化并不是必需的。进一步的研究发现，在MDA-MB-231这一恶化程度较高的乳腺癌细胞中，细胞膜上并没有PHB1的表达，而在MCF-7这一恶化程度比较低的乳腺癌细胞及血管内皮细胞HUVEC中，细胞膜上有PHB1的表达，因此在我们看来，PHB1在MDA-MB-231中的表达可能是异常的。在HUVEC中，通过RNA干扰降低PHB1的表达后，PAR1的激动型内化程度降低，而基础型内化没有受到影响。并且PAR1介导的Erk1/2的磷酸化时间延长到15分钟，而对照只到10分钟。同时，在MDA-MB-231中，PAR1的激动型内化程度很低，并且它的Erk1/2磷酸化很高，持续时间很长。因此，我们认为可能是PHB1参与调节了HUVEC中的PAR1的激动型内化，而由于在MDA-MB-231的细胞膜上没有内源性的PHB1的表达，从而无法调节PAR1的激动型内化，可能是MDA-MB-231中PAR1持续活化的原因之一。同时，在MDA-MB-231中，通过RNA干扰降低PHB1的表达后，PAR1激活后的降解程度变高，而在HUVEC中不存在这种现象。因此，我们认为，正是由于PHB1在MDA-MB-231中的功能异常，导致了PAR1的功能异常，主要是不转运、不降解，表现为PAR1的持续活化，从而使MDA-MB-231具有高浸润的特性。如果可以对PHB1进行专一调控，可能使乳腺癌细胞的浸润性降低，从而可以达到治疗的目的。 |
| 英文摘要 | Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs are reported to be involved in typhoid fever, obesity and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown. In our present study, PHB1 and PHB2 were detected on the surface of human platelets using flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease-activated receptor 1 (PAR1), as determined by Bm-TFF2 (a PAR1 agonist)-affinity chromatography, co-immunoprecipitation and confocal microscopy. The platelet aggregation, ?IIb?3 activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U/ml) or PAR1-activating peptide (PAR1-AP, 20 ?M) were reduced or abolished as a result of the blockade of PHBs by anti-PHB antibodies or their Fab fragments; however, the same results were not observed when induced by high concentrations of thrombin (0.6 U/ml) or PAR4-AP (300 ?M). The calcium mobilization in MEG-01 megakaryocytes stimulated by PAR1-AP was significantly suppressed by PHB depletion using RNA interference against PHB1 and PHB2. Until recently, PHBs were unknown regulators of PAR1 signaling and may be effective targets for anti-platelet therapy. PHB1 is a multifunctional protein and is related with many disease, however, there is no inhibitor or antagonist of PHB1 was founded. So we find a peptide (Pep5) in a five-amino-acid peptide library of all possible pentapeptide (205 = 3.2 million), which has the same motif with proteins associate with PHB1, based on the structure of prohibitin. According to our experiment data, we found that Pep5 could interact with PHB1 by Pep5-affinity chromatography and molecular docking. Besides, we found that Pep5 could inhibit PAR1-AP and low concentration of thrombin induced platelet aggregation and calcium mobilization at the concentration of 300????which IC50 is 200????????, and has no effect on PAR4-AP and collagen induced platelet aggregation. In this study, we found PHB1 could certainly be the target of antiplatelet therapy, Pep5 could be an antagonist of PHB1 and necessary modification of Pep5 could improve its efficiency. PHB1 is involved in PAR1-mediated platelet aggregation and is necessary for PAR1 related signalling pathway activation in human platelets and MEG-01. However, its function in PAR1 desensitization, trafficking and degradation is unknown. In HUVEC and MDA-MB-231 cells, PHB1 does not participate in PAR1-mediated signalling activation, PHB depletion using RNA interference against PHB1 does not affect PAR1-mediated Ca2+ mobilization in these two kinds of cell lines. However, whether it regulates the termination process of receptor signalling, such as desensilization, internalization and degradation, is unknown. In this section, we found that in HUVEC, PHB1 colocalized with PAR1 on the plasma membrane, in MDA-MB-231 cells, PHB1 dose not express on the plasma membrane. In HUVEC, depletion PHB1 abolished activated PAR1 internalization but not its constitutive internalization. Besides, the phosphorylation of Erk1/2 extended to 15min in PHB1 depleted HUVEC cells, while the control is just 10min. In MDA-MB-231, the PAR1 signaling is durable when it was activated. Our results show that the level of PAR1 activated internalization in this cell is very low, the phosphorylation of Erk1/2 is chronic, which may be responsible for PAR1 persistent signalling activation. Also, when depleted PHB1 in MDA-MB-231, the degradation of activated PAR1 is increased, while this is not found in HUVEC. hence we deduced that the expression of PHB1 in MDA-MB-231 is abnormal, maybe it can be the target to cure breast cancer. |
| 语种 | 中文 |
| 公开日期 | 2012-12-12 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7134]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 王严戒. 抑素蛋白对蛋白酶激活受体１的分子调控机制研究[D]. 北京. 中国科学院大学. 2012. |
入库方式： OAI收割
来源：昆明动物研究所
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