微囊藻毒素LR刺激HEK293細胞產(chǎn)生的PP2A調節(jié)機制及其對細胞凋亡命運的影響
發(fā)布時間：2018-08-05 20:32
【摘要】：水體富營養(yǎng)化以及由此造成的藍藻爆發(fā)是當今人類面臨的嚴重問題。這其中微囊藻屬藻爆發(fā)引起的水體微囊藻毒素(MCs, microcystins)污染對人類的健康的影響尤其令人關注。例如,微囊藻毒素LR(MCLR, microcystin-LR)具有肝毒性、腎毒性、神經(jīng)毒性等,且是毒性最強的微囊藻毒素之一。研究顯示,MCLR既能造成細胞凋亡,又具有促腫瘤作用。然而,細胞暴露于MCLR后,其命運的決定因素尚待揭示。 蛋白磷酸酶2A(PP2A, protein phosphatase2A)是MCLR在細胞內的主要靶點。PP2A在細胞內具有重要的作用,參與幾乎所有細胞生理活動,包括細胞增殖、細胞代謝、細胞分化和轉變、DNA修復、細胞凋亡等。PP2A全酶由結構亞基(PP2A/A)、活性亞基(PP2A/C)以及調節(jié)亞基(PP2A/B)組成。其中,調節(jié)亞基決定PP2A全酶的下游底物、亞細胞定位以及具體的生理功能。目前已發(fā)現(xiàn)有75種PP2A全酶,各全酶都具有眾多的底物。此外,還有一小部分PP2A/C亞基與a4蛋白結合但只具有較低的活性。這一結合狀態(tài)與細胞在應激狀態(tài)下對PP2A活性的調節(jié)具有重要的關聯(lián)。 已有研究表明,MCLR直接與PP2A的活性亞基(PP2A/C)結合而造成的PP2A活性損失是MCLR造成細胞損傷的重要機制。但是,除開直接與PP2A/C結合以抑制其活性之外,MCLR是否影響PP2A其他亞基,以及MCLR如何具體影響細胞內PP2A全酶的活性則尚不明晰。此外,因為PP2A具有重要的生理功能,細胞在應激狀態(tài)下對于PP2A的活性具有密切的調控,例如PP2A/C磷酸化、甲基化等翻譯后修飾；產(chǎn)生神經(jīng)酰胺后激活一部分PP2A(CAPP, ceramide activated protein phosphatase);α4蛋白與低活性PP2A解離以代償活性損失等。由此我們提問：細胞應對MCLR的影響會產(chǎn)生哪些調節(jié)機制；這些調節(jié)機制是否足夠代償MCLR對細胞內PP2A的活性抑制作用,以及隨之會產(chǎn)生哪些細胞學效應? 根據(jù)前期實驗的蛋白質組學研究發(fā)現(xiàn),細胞暴露于MC后,細胞內眾多信號蛋白發(fā)生改變,并且這些信號蛋白大多與PP2A相關。由此本研究假設,細胞暴露于MCLR后,細胞內PP2A的活性,尤其是其全酶的活性,以及細胞對PP2A的調節(jié)機制,對于細胞命運的決定具有重要的作用。本研究選取人胚腎細胞系(HEK293, Human Embryonic Kidney293)這一腎臟來源的細胞作為研究對象,運用免疫印跡、免疫共沉淀、免疫熒光等方法,研究在MCLR對其細胞活力沒有嚴重致死效應的條件下,HEK293細胞內PP2A亞基水平和活性的變化、PP2A底物磷酸化水平、PP2A活性調節(jié)機制、細胞骨架和細胞黏連蛋白的形態(tài)、細胞命運的選擇。此外,本研究還選取小鼠作為活體研究對象,驗證MCLR的腎臟毒性。 主要結果： 1. MCLR直接與HEK293細胞內PP2A/C亞基結合。在本實驗濃度下,MCLR對細胞活力有下調的趨勢,但并無嚴重的抑制效應。 2. MCLR不影響細胞內PP2A/A, PP2A/C和PP2A/B56δ蛋白水平,但上調PP2A/B55α和PP2A/B56α蛋白水平；MCLR下調細胞內PP2A/C甲基化,但不影響其磷酸化；MCLR引起PP2A與其泛素連接酶Mid1解離；高濃度MCLR造成PP2A/A和PP2A/C部分解離；MCLR引起PP2A/B55α部分聚集于高爾基體,但對PP2A/C和PP2A/B56α亞基定位影響不明顯；MCLR引起細胞內PP2A/C和α4蛋白解離,并且造成α4蛋白定位于細胞核。 3. MCLR對細胞內PP2A總體活性影響呈低濃度刺激活性、高濃度抑制活性。 4. MCLR引起HEK293細胞生成神經(jīng)酰胺。用神經(jīng)酰胺合成酶抑制劑DESI共處理細胞后,MCLR引起的PP2A/B55α和PP2A/B56α蛋白水平上調效應消除；低濃度MCLR引起PP2A活性上調效應消除；高濃度MCLR對PP2A的活性抑制作用增強,以致PP2A活性幾乎完全被抑制。 5. MCLR引起PP2A/B56a全酶的下游底物c-Myc磷酸化降低,但不影響其蛋白水平；PP2A/B56a全酶下游底物Bad蛋白水平升高,磷酸化比例降低。MCLR還引起凋亡相關蛋白Bcl-2蛋白水平降低,但不影響B(tài)ax蛋白水平。與DESI共處理后,MCLR對Bcl-2和Bad蛋白水平的改變減弱。MCLR還引起p38MAPK以及JNK蛋白磷酸化上調。 6. MCLR引起HEK293細胞形態(tài)改變。MCLR引起細胞微絲蛋白解聚,中間纖維之一的波形蛋白和微管蛋白聚縮,這一現(xiàn)象與神經(jīng)酰胺處理HEK293細胞后細胞骨架的改變相似。與DESI共處理后,MCLR對細胞骨架的改變減弱。MCLR還引起骨架相關蛋白Rac1和Mid1定位于細胞核。 7. MCLR引起HEK293細胞粘著斑蛋白形態(tài)改變,并引起細胞貼壁能力減弱。MCLR引起細胞核聚縮化和片段化,引起細胞凋亡。與DESI共處理后,MCLR對細胞貼壁的影響以及刺激細胞凋亡的效應減弱。 8.小鼠腹腔注射MCLR毒素后,腎臟可檢測神經(jīng)酰胺的生成,并可檢測到升高的細胞凋亡。 主要結論： MCLR不但能直接與HEK293細胞內PP2A結合,還能引起細胞對PP2A的調節(jié)作用,包括產(chǎn)生神經(jīng)酰胺,PP2A/C與α4蛋白解離等。此外,MCLR對細胞骨架、細胞貼壁以及細胞凋亡產(chǎn)生的影響與神經(jīng)酰胺相關,并且與α4蛋白與PP2A/C解離后失去原有功能的推論相符合。MCLR還能刺激小鼠腎臟產(chǎn)生神經(jīng)酰胺并產(chǎn)生細胞凋亡。本實驗結果顯示PP2A全酶活性及細胞對PP2A的調節(jié)作用對于細胞暴露于MCLR的凋亡命運的決定具有重要的作用。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R114
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相關重要報紙文章 前10條
1 張?zhí)锟?細胞調亡的意義[N];中國人口報;2002年
2 記者 張Z，
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