【作者】 李覃；

【导师】 徐立红；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 水体富营养化以及由此造成的蓝藻爆发是当今人类面临的严重问题。这其中微囊藻属藻爆发引起的水体微囊藻毒素(MCs, microcystins)污染对人类的健康的影响尤其令人关注。例如,微囊藻毒素LR(MCLR, microcystin-LR)具有肝毒性、肾毒性、神经毒性等,且是毒性最强的微囊藻毒素之一。研究显示,MCLR既能造成细胞凋亡,又具有促肿瘤作用。然而,细胞暴露于MCLR后,其命运的决定因素尚待揭示。蛋白磷酸酶2A(PP2A, protein phosphatase2A)是MCLR在细胞内的主要靶点。PP2A在细胞内具有重要的作用,参与几乎所有细胞生理活动,包括细胞增殖、细胞代谢、细胞分化和转变、DNA修复、细胞凋亡等。PP2A全酶由结构亚基(PP2A/A)、活性亚基(PP2A/C)以及调节亚基(PP2A/B)组成。其中,调节亚基决定PP2A全酶的下游底物、亚细胞定位以及具体的生理功能。目前已发现有75种PP2A全酶,各全酶都具有众多的底物。此外,还有一小部分PP2A/C亚基与a4蛋白结合但只具有较低的活性。这一结合状态与细胞在应激状态下对PP2A活性的调节具有重要的关联。已有研究表明,MCLR直接与PP2A的活性亚基(PP2A/C)结合而造成的PP2A活性损失是MCLR造成细胞损伤的重要机制。但是,除开直接与PP2A/C结合以抑制其活性之外,MCLR是否影响PP2A其他亚基,以及MCLR如何具体影响细胞内PP2A全酶的活性则尚不明晰。此外,因为PP2A具有重要的生理功能,细胞在应激状态下对于PP2A的活性具有密切的调控,例如PP2A/C磷酸化、甲基化等翻译后修饰；产生神经酰胺后激活一部分PP2A(CAPP, ceramide activated protein phosphatase);α4蛋白与低活性PP2A解离以代偿活性损失等。由此我们提问：细胞应对MCLR的影响会产生哪些调节机制；这些调节机制是否足够代偿MCLR对细胞内PP2A的活性抑制作用,以及随之会产生哪些细胞学效应?根据前期实验的蛋白质组学研究发现,细胞暴露于MC后,细胞内众多信号蛋白发生改变,并且这些信号蛋白大多与PP2A相关。由此本研究假设,细胞暴露于MCLR后,细胞内PP2A的活性,尤其是其全酶的活性,以及细胞对PP2A的调节机制,对于细胞命运的决定具有重要的作用。本研究选取人胚肾细胞系(HEK293, Human Embryonic Kidney293)这一肾脏来源的细胞作为研究对象,运用免疫印迹、免疫共沉淀、免疫荧光等方法,研究在MCLR对其细胞活力没有严重致死效应的条件下,HEK293细胞内PP2A亚基水平和活性的变化、PP2A底物磷酸化水平、PP2A活性调节机制、细胞骨架和细胞黏连蛋白的形态、细胞命运的选择。此外,本研究还选取小鼠作为活体研究对象,验证MCLR的肾脏毒性。主要结果：1. MCLR直接与HEK293细胞内PP2A/C亚基结合。在本实验浓度下,MCLR对细胞活力有下调的趋势,但并无严重的抑制效应。2. MCLR不影响细胞内PP2A/A, PP2A/C和PP2A/B56δ蛋白水平,但上调PP2A/B55α和PP2A/B56α蛋白水平；MCLR下调细胞内PP2A/C甲基化,但不影响其磷酸化；MCLR引起PP2A与其泛素连接酶Mid1解离；高浓度MCLR造成PP2A/A和PP2A/C部分解离；MCLR引起PP2A/B55α部分聚集于高尔基体,但对PP2A/C和PP2A/B56α亚基定位影响不明显；MCLR引起细胞内PP2A/C和α4蛋白解离,并且造成α4蛋白定位于细胞核。3. MCLR对细胞内PP2A总体活性影响呈低浓度刺激活性、高浓度抑制活性。4. MCLR引起HEK293细胞生成神经酰胺。用神经酰胺合成酶抑制剂DESI共处理细胞后,MCLR引起的PP2A/B55α和PP2A/B56α蛋白水平上调效应消除；低浓度MCLR引起PP2A活性上调效应消除；高浓度MCLR对PP2A的活性抑制作用增强,以致PP2A活性几乎完全被抑制。5. MCLR引起PP2A/B56a全酶的下游底物c-Myc磷酸化降低,但不影响其蛋白水平；PP2A/B56a全酶下游底物Bad蛋白水平升高,磷酸化比例降低。MCLR还引起凋亡相关蛋白Bcl-2蛋白水平降低,但不影响Bax蛋白水平。与DESI共处理后,MCLR对Bcl-2和Bad蛋白水平的改变减弱。MCLR还引起p38MAPK以及JNK蛋白磷酸化上调。6. MCLR引起HEK293细胞形态改变。MCLR引起细胞微丝蛋白解聚,中间纤维之一的波形蛋白和微管蛋白聚缩,这一现象与神经酰胺处理HEK293细胞后细胞骨架的改变相似。与DESI共处理后,MCLR对细胞骨架的改变减弱。MCLR还引起骨架相关蛋白Rac1和Mid1定位于细胞核。7. MCLR引起HEK293细胞粘着斑蛋白形态改变,并引起细胞贴壁能力减弱。MCLR引起细胞核聚缩化和片段化,引起细胞凋亡。与DESI共处理后,MCLR对细胞贴壁的影响以及刺激细胞凋亡的效应减弱。8.小鼠腹腔注射MCLR毒素后,肾脏可检测神经酰胺的生成,并可检测到升高的细胞凋亡。主要结论：MCLR不但能直接与HEK293细胞内PP2A结合,还能引起细胞对PP2A的调节作用,包括产生神经酰胺,PP2A/C与α4蛋白解离等。此外,MCLR对细胞骨架、细胞贴壁以及细胞凋亡产生的影响与神经酰胺相关,并且与α4蛋白与PP2A/C解离后失去原有功能的推论相符合。MCLR还能刺激小鼠肾脏产生神经酰胺并产生细胞凋亡。本实验结果显示PP2A全酶活性及细胞对PP2A的调节作用对于细胞暴露于MCLR的凋亡命运的决定具有重要的作用。更多还原

【Abstract】 Eutrophication and blue alga bloom in waters are causing serious environmental disasters worldwide. One group of blue alga, microcystis, produces microcystins (MCs) in water bloom that pose severe hazard to human health due to their strong toxicity. Among them, microcystin-LR (MCLR) has hepatoxicity, nephrotoxicity and neurotoxicity, and is one of the most toxic member of MCs. Studies have revealed that MCLR can cause cell death, while can also promote carcinogenesis. However, under exposure of MCLR, the determinant factors of cell fate are not clear.Protein phosphatase2A (PP2A) is a main target of MCLR. PP2A has essentially important functions in cells and is involved in almost all the cellular activities, such as cell proliferation, metabolism, differentiation, transformation, DNA repair and apoptosis. PP2A holoenzyme is comprised of a scaffold subunit (PP2A/A), a catalytic subunit (PP2A/C), and one of many regulatory subunits (PP2A/B). The regulatory subunit determines the substrates, sub-cellular localization and the function of PP2A holoenzyme. At least75kinds of PP2A holoenzymes have been found and each has a variety of substrates. Other than the classical combination, a small part of PP2A/C binds to a4protein and has lower activity than the other forms. However, this form of combination plays an important role in cellular regulation of PP2A activity when under stress. Previous studies have found that the direct binding of MCLR to PP2A/C inhibits its phosphatase activity. This feature of MCLR has long been considered the major mechanism by which MCLR poses cytotoxicity. However, apart from the direct binding and thus inhibition of PP2A activity, other effects of MCLR on cellular PP2A holoenzymes are largely unknown. Moreover, because of the essential importance of PP2A, its activity is under tight regulation by the cells. So far, it has been found that the cells regulate PP2A activity by phosphorylation and methylation of PP2A/C, by generating ceramide which upregulates certain forms of PP2A holoenzyme (CAPP, ceramide activated protein phosphatase), as well as by dissociation of PP2A/C and α4protein. Therefore we ask, when under MCLR insult, by which mechanisms cells may regulate PP2A activity; is the regulatory effect enough to compensate the activity loss caused by MCLR; and what are the consequential cellular effects?Previous proteomic studies have shown that under the exposure of MC, a large spectrum of signal proteins are altered. Importantly, most of those proteins are related to PP2A. Thus, we hypothesize that under the exposure of MCLR, the activity of PP2A, especially the activity of its holoenzyme, as well as the regulation of PP2A from the cells, play an important role in the fate determination of the cells. The present study utilizes Human Embryonic Kidney293(HEK293) cell line, to study the PP2A subunit and activity change, the phosphorylation of its substrates, the regulation of PP2A activity, the morphological change of cytoskeleton and focal adhesion, and cell fate, at the time when cells are treated with MCLR at concentrations that do not cause massive cell death. This study also uses mouse kidney as in vivo subject to reinforce some in vitro result.Major findings1. MCLR can directly bind to PP2A/C in HEK293cells. The concentrations of MCLR used in this study do not cause massive cell death, though tend to inhibit their viability.2. MCLR does not affect the protein levels of PP2A/A, PP2A/C and PP2A/B56δ in HEK293cells, but upregulates the protein levels of PP2A/B55a and PP2A/B56a; MCLR downregulates the methylation and ubiquitination of PP2A/C, but does not affect its phosphorylation; MCLR causes PP2A/C dissociate from its ubiquitinligase Mid1; relative higher concentration of MCLR causes dissociation of PP2A/A and PP2A/C; MCLR causes PP2A/B55a locate to Golgi apparatus but not PP2A/B56a; MCLR causes the dissociation of PP2A/C and a4protein, and a4protein locate to nucleus, but not PP2A/C.3. Relatively low concentrations of MCLR upregulate, while relatively high concentrations of MCLR inhibit cellular PP2A activity.4. MCLR induces the generation of ceramide. Using desipramine (DESI) to inhibit the generation of ceramide can alleviate the upregulation of PP2A/B55a and PP2A/B56α on protein level, and the upregualtion of PP2A activity at relatively low MCLR concentration, while aggravate the inhibition of PP2A by relatively high concentration of MCLR treatment, till almost complete inhibition.5. MCLR decreases of phosphorylation of c-Myc, a substrate of PP2A holoenzyme containing PP2A/B56a, while does not affect c-Myc protein level. MCLR upregulates the protein level, and decreases of phosphorylation ratio of Bad, another substrate of PP2A holoenzyme containing PP2A/B56a. MCLR also downregulates Bcl-2on protein level, but does not affect Bax. Under DESI co-treatment, the effects of MCLR on Bad and Bcl-2are abated. MCLR also upregulates the phosphorylation of p38MAPK and JNK.6. MCLR changes the morphology of HEK293cells. MCLR causes the depolymerization of cell filamentsactin, and the contraction of vimentin and microtubulin. The phenotype is similar to cells treated only with C6-cereamide. The disruption of cytoskeleton is alleviated when co-treated with MCLR and DESI. MCLR also causes cytoskeleton-related proteins Racl and Midi locate to nucleus.7. MCLR changes the morphology of viculin and disrupts the cell attachment to culture matrix. MCLR causes condensation and fragmentation of nuclei, indicating apoptosis. These effects are aborted when co-treated with DESI.8. Ceramide generation and increased apoptosis can be found in mouse kidney after MCLR injection. Major conclusionBesides that MCLR can directly bind to PP2A in HEK293cells, MCLR can also stimulate regulatory of PP2A in the cells, including the generation of ceramide, as well as dissociation of PP2A/C and a4. Furthermore, MCLR affects the cytoskeleton stability, cell attachment and induces apoptosis of HEK293cells. These cellular effects are related with ceramide, and also consistent with the loss of a4function due to dissociation with PP2A/C. Moreover, MCLR can induce ceramide generation and apoptosis in mouse kidney. Therefore, the results suggest that the holoenzyme activity of PP2A, and cellularregulation of PP2A, may be essential for the apoptotic fate of HEK293cells under MCLR exposure.更多还原