微囊藻毒素LR刺激HEK293细胞产生的PP2A调节机制及其对细胞凋亡命运的影响
发布时间：2018-08-05 20:32
【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。
[Abstract]:The eutrophication of water bodies and the resulting cyanobacteria are serious problems faced by humans today. The effects of microcystin (MCs, microcystins) on human health are particularly concerned. For example, the microcystin LR (MCLR, microcystin-LR) has hepatotoxicity, nephrotoxicity, and nerve. Toxicity and so on, and one of the most toxic microcystins. Studies have shown that MCLR can cause both apoptosis and tumor promoting effect. However, the determinants of their fate have yet to be revealed after the cells are exposed to MCLR.
Protein phosphatase 2A (PP2A, protein phosphatase2A) is the major target of MCLR in cells,.PP2A, which plays an important role in cells. It participates in almost all cell physiological activities, including cell proliferation, cell metabolism, cell differentiation and transformation, DNA repair, apoptosis and other.PP2A total enzymes from structural subunits (PP2A/A), active subunits (PP2A/C) and modulation. The subunit (PP2A/B) consists of a subunit that regulates the downstream substrates, subcellular localization and specific physiological functions of the PP2A whole enzyme. 75 PP2A total enzymes have been found and all enzymes have numerous substrates. In addition, a small portion of the PP2A/C subunit is associated with the A4 protein but only has a lower activity. There is an important correlation between the regulation of PP2A activity under stress.
It has been shown that the loss of PP2A activity caused by the binding of MCLR directly with the active subunit of PP2A (PP2A/C) is an important mechanism for cell damage caused by MCLR. However, it is not clear whether MCLR affects other subunits of PP2A, and how MCLR affects the activity of PP2A total enzyme in cells. In addition, because PP2A has important physiological functions, cells regulate the activity of PP2A closely in stress state, such as PP2A/C phosphorylation, methylation and post-translational modification; after producing ceramide, it activates a part of PP2A (CAPP, ceramide activated protein phosphatase), and the dissociation of alpha 4 protein and low activity PP2A is compensatory activity loss. In this case, we ask: what regulatory mechanisms are produced by the cell response to the MCLR; are these regulatory mechanisms sufficient to compensate for the inhibitory effect of MCLR on intracellular PP2A activity, and what cytological effects will be produced accordingly?
According to the proteomics study of the earlier experiment, it was found that after the cells were exposed to MC, many of the signal proteins in the cells changed, and most of these proteins were associated with PP2A. This study assumed that after the cells were exposed to MCLR, the activity of intracellular PP2A, especially the activity of its whole enzyme, and the regulation mechanism of cell to PP2A, were used for cells. The determination of fate plays an important role. In this study, the cells of HEK293 (Human Embryonic Kidney293), a kidney source, were selected as the research object. Immunoblotting, immunofluorescence, immunofluorescence and other methods were used to study the PP2A subunit in HEK293 cells under the condition that MCLR had no serious lethal effect on the cell viability. The changes in basal level and activity, the phosphorylation level of PP2A substrate, the regulatory mechanism of PP2A activity, the morphology of cytoskeleton and cell mucin, and the selection of cell fate. In addition, this study also selected mice as a living object to verify the renal toxicity of MCLR.
Main results:
1. MCLR binds directly to PP2A/C subunit in HEK293 cells. At this concentration, MCLR has a downward trend in cell viability, but has no serious inhibitory effect.
2. MCLR did not affect the level of intracellular PP2A/A, PP2A/C and PP2A/B56 delta protein, but up regulation of PP2A/B55 alpha and PP2A/B56 alpha protein; MCLR downregulated intracellular PP2A/C methylation, but did not affect its phosphorylation; MCLR caused PP2A and its ubiquitin ligase dissociation; high concentration MCLR resulted in partial dissociation and partial dissociation. In Golgi body, there is no obvious effect on the localization of PP2A/C and PP2A/B56 alpha subunits. MCLR causes the dissociation of intracellular PP2A/C and alpha 4 protein, and the alpha 4 protein is located in the nucleus.
3. the effect of MCLR on the overall activity of PP2A in cells showed low concentration activity and high inhibitory activity.
4. MCLR caused HEK293 cells to produce ceramide. After the cells were co treated with ceramide synthase inhibitor DESI, the up-regulation effect of MCLR induced PP2A/B55 alpha and PP2A/B56 alpha protein was eliminated; the low concentration MCLR caused the up regulation effect of PP2A activity to be eliminated; high concentration MCLR enhanced the activity of PP2A, so that PP2A activity was almost completely completely activated. Inhibition.
5. MCLR decreased the phosphorylation of the downstream substrate c-Myc of the PP2A/B56a whole enzyme, but did not affect its protein level; the level of Bad protein in the downstream substrate of PP2A/B56a was elevated, the ratio of phosphorylation to.MCLR also caused the decrease of the apoptosis related protein Bcl-2 protein level, but did not affect the level of Bax protein. After CO processing with DESI, MCLR was to Bcl-2 and Bad protein levels. The altered.MCLR weakened the phosphorylation of p38MAPK and JNK protein.
6. MCLR caused the morphologic change of HEK293 cells to cause the cell microprotein depolymerization, the vimentin and microtubulin condensation in one of the intermediate fibers, this phenomenon was similar to the change of the cytoskeleton after the ceramide treated the HEK293 cells. After CO processing with DESI, the modification of the cytoskeleton of MCLR weakened.MCLR and caused the skeleton related protein Rac1 and Mi. D1 is located in the nucleus.
7. MCLR caused the morphologic changes of the adhesion protein of HEK293 cells, and caused the weakening of cell adhesion ability by.MCLR, which caused cell nuclear polycondensation and fragmentation and caused cell apoptosis. After CO processing with DESI, the effect of MCLR on cell adhesion and the effect of stimulating cell apoptosis was weakened.
8. after the intraperitoneal injection of MCLR toxin, the kidney could detect ceramide production and detect increased apoptosis.
The main conclusions are as follows:
MCLR can not only directly bind to PP2A in HEK293 cells, but also induce the regulation of cell to PP2A, including the production of ceramide, PP2A/C and alpha 4 protein dissociation. In addition, the effect of MCLR on the cytoskeleton, cell adhesion and apoptosis is related to ceramide, and the inference of the loss of the original function after the dissociation of alpha 4 protein and PP2A/C .MCLR can also stimulate the production of ceramide and induce apoptosis in the kidney of mice. The results of this experiment show that the PP2A total enzyme activity and the regulation of cell to PP2A play an important role in determining the apoptosis fate of MCLR.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
[Abstract]:The eutrophication of water bodies and the resulting cyanobacteria are serious problems faced by humans today. The effects of microcystin (MCs, microcystins) on human health are particularly concerned. For example, the microcystin LR (MCLR, microcystin-LR) has hepatotoxicity, nephrotoxicity, and nerve. Toxicity and so on, and one of the most toxic microcystins. Studies have shown that MCLR can cause both apoptosis and tumor promoting effect. However, the determinants of their fate have yet to be revealed after the cells are exposed to MCLR.
Protein phosphatase 2A (PP2A, protein phosphatase2A) is the major target of MCLR in cells,.PP2A, which plays an important role in cells. It participates in almost all cell physiological activities, including cell proliferation, cell metabolism, cell differentiation and transformation, DNA repair, apoptosis and other.PP2A total enzymes from structural subunits (PP2A/A), active subunits (PP2A/C) and modulation. The subunit (PP2A/B) consists of a subunit that regulates the downstream substrates, subcellular localization and specific physiological functions of the PP2A whole enzyme. 75 PP2A total enzymes have been found and all enzymes have numerous substrates. In addition, a small portion of the PP2A/C subunit is associated with the A4 protein but only has a lower activity. There is an important correlation between the regulation of PP2A activity under stress.
It has been shown that the loss of PP2A activity caused by the binding of MCLR directly with the active subunit of PP2A (PP2A/C) is an important mechanism for cell damage caused by MCLR. However, it is not clear whether MCLR affects other subunits of PP2A, and how MCLR affects the activity of PP2A total enzyme in cells. In addition, because PP2A has important physiological functions, cells regulate the activity of PP2A closely in stress state, such as PP2A/C phosphorylation, methylation and post-translational modification; after producing ceramide, it activates a part of PP2A (CAPP, ceramide activated protein phosphatase), and the dissociation of alpha 4 protein and low activity PP2A is compensatory activity loss. In this case, we ask: what regulatory mechanisms are produced by the cell response to the MCLR; are these regulatory mechanisms sufficient to compensate for the inhibitory effect of MCLR on intracellular PP2A activity, and what cytological effects will be produced accordingly?
According to the proteomics study of the earlier experiment, it was found that after the cells were exposed to MC, many of the signal proteins in the cells changed, and most of these proteins were associated with PP2A. This study assumed that after the cells were exposed to MCLR, the activity of intracellular PP2A, especially the activity of its whole enzyme, and the regulation mechanism of cell to PP2A, were used for cells. The determination of fate plays an important role. In this study, the cells of HEK293 (Human Embryonic Kidney293), a kidney source, were selected as the research object. Immunoblotting, immunofluorescence, immunofluorescence and other methods were used to study the PP2A subunit in HEK293 cells under the condition that MCLR had no serious lethal effect on the cell viability. The changes in basal level and activity, the phosphorylation level of PP2A substrate, the regulatory mechanism of PP2A activity, the morphology of cytoskeleton and cell mucin, and the selection of cell fate. In addition, this study also selected mice as a living object to verify the renal toxicity of MCLR.
Main results:
1. MCLR binds directly to PP2A/C subunit in HEK293 cells. At this concentration, MCLR has a downward trend in cell viability, but has no serious inhibitory effect.
2. MCLR did not affect the level of intracellular PP2A/A, PP2A/C and PP2A/B56 delta protein, but up regulation of PP2A/B55 alpha and PP2A/B56 alpha protein; MCLR downregulated intracellular PP2A/C methylation, but did not affect its phosphorylation; MCLR caused PP2A and its ubiquitin ligase dissociation; high concentration MCLR resulted in partial dissociation and partial dissociation. In Golgi body, there is no obvious effect on the localization of PP2A/C and PP2A/B56 alpha subunits. MCLR causes the dissociation of intracellular PP2A/C and alpha 4 protein, and the alpha 4 protein is located in the nucleus.
3. the effect of MCLR on the overall activity of PP2A in cells showed low concentration activity and high inhibitory activity.
4. MCLR caused HEK293 cells to produce ceramide. After the cells were co treated with ceramide synthase inhibitor DESI, the up-regulation effect of MCLR induced PP2A/B55 alpha and PP2A/B56 alpha protein was eliminated; the low concentration MCLR caused the up regulation effect of PP2A activity to be eliminated; high concentration MCLR enhanced the activity of PP2A, so that PP2A activity was almost completely completely activated. Inhibition.
5. MCLR decreased the phosphorylation of the downstream substrate c-Myc of the PP2A/B56a whole enzyme, but did not affect its protein level; the level of Bad protein in the downstream substrate of PP2A/B56a was elevated, the ratio of phosphorylation to.MCLR also caused the decrease of the apoptosis related protein Bcl-2 protein level, but did not affect the level of Bax protein. After CO processing with DESI, MCLR was to Bcl-2 and Bad protein levels. The altered.MCLR weakened the phosphorylation of p38MAPK and JNK protein.
6. MCLR caused the morphologic change of HEK293 cells to cause the cell microprotein depolymerization, the vimentin and microtubulin condensation in one of the intermediate fibers, this phenomenon was similar to the change of the cytoskeleton after the ceramide treated the HEK293 cells. After CO processing with DESI, the modification of the cytoskeleton of MCLR weakened.MCLR and caused the skeleton related protein Rac1 and Mi. D1 is located in the nucleus.
7. MCLR caused the morphologic changes of the adhesion protein of HEK293 cells, and caused the weakening of cell adhesion ability by.MCLR, which caused cell nuclear polycondensation and fragmentation and caused cell apoptosis. After CO processing with DESI, the effect of MCLR on cell adhesion and the effect of stimulating cell apoptosis was weakened.
8. after the intraperitoneal injection of MCLR toxin, the kidney could detect ceramide production and detect increased apoptosis.
The main conclusions are as follows:
MCLR can not only directly bind to PP2A in HEK293 cells, but also induce the regulation of cell to PP2A, including the production of ceramide, PP2A/C and alpha 4 protein dissociation. In addition, the effect of MCLR on the cytoskeleton, cell adhesion and apoptosis is related to ceramide, and the inference of the loss of the original function after the dissociation of alpha 4 protein and PP2A/C .MCLR can also stimulate the production of ceramide and induce apoptosis in the kidney of mice. The results of this experiment show that the PP2A total enzyme activity and the regulation of cell to PP2A play an important role in determining the apoptosis fate of MCLR.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114
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