锌离子对蛋白磷酸酯酶2A的调节及其在阿尔茨海默病中的作用

发布时间：2018-06-04 22:09

本文选题：锌 + 蛋白磷酸酶　；参考：《华中科技大学》2013年博士论文

【摘要】：阿尔茨海默病（Alzheimer’s Disease, AD）是最常见的老年性痴呆症，其主要病理学改变是脑内神经元纤维缠结（NFTs）和老年斑(SP)的形成。NFTs的主要成分是过度磷酸化的微管相关蛋白tau,异常磷酸化的tau丧失其促微管组装和稳定微管的功能,并聚集成为成对螺旋丝（PHF），最终导致NFT的形成、突触的退变和神经元的丢失,在AD的发生、发展中起关键作用。而tau的磷酸化主要受相关蛋白激酶（protein kinases, PK）和蛋白磷酸酯酶（protein phosphatases,PP）的调节。大量的研究事实表明，蛋白磷酸酯酶2A(PP2A)的活性在阿尔茨海默病人脑中下降并参与AD样tau蛋白异常磷酸化的形成，但导致PP2A活性下降的上游因素和机制尚未阐明。锌（Zn）离子，是脑内含量较为丰富的微量元素之一，参与神经传导，抗氧化反应，并调节多种酶和生长因子的活性。大量研究结果表明，胞外锌离子水平增高可导致多种蛋白质发生酪氨酸磷酸化修饰。前期研究结果显示PP2A催化亚单位酪氨酸307位点（Y307）磷酸化后可明显抑制PP2A活性，而在AD易感脑区锌离子水平增高，提示锌离子可能参与了AD脑内PP2A活性的下调而促进tau蛋白过度磷酸化。且到目前为止Zn对PP2A的调节及机制尚无报道。我们在本研究中重点探讨了锌离子对PP2A的调节及其在tau过度磷酸化中的作用，并且从分子水平解释了Zn对PP2A的直接和间接抑制作用机制。主要结果如下：第一部分锌离子通过激活Src途径抑制PP2A活性和导致tau蛋白过度磷酸化蛋白磷酸酯酶2A(PP2A)的活性在阿尔茨海默病（AD）人脑中下降并参与AD样tau蛋白异常磷酸化的形成，但导致PP2A活性下降的上游因素和机制尚未阐明。有文献报道显示，PP2A催化亚基的酪氨酸307位点（Y307）磷酸化修饰是导致PP2A失活的直接原因，但何种机制促使PP2A发生失活性修饰还不清楚。大量研究结果表明，胞外锌离子水平增高可导致多种蛋白质发生酪氨酸磷酸化修饰，而在AD易感脑区锌离子水平增高，提示锌离子可能参与了AD脑内PP2A活性的下调而促进tau蛋白过度磷酸化。【目的】阐明锌离子是否通过调节PP2A活性促进tau蛋白异常过度磷酸化及调节PP2A活性的机制。【材料和方法】实验在整体和细胞水平进行。整体水平采用雄性SD大鼠，侧脑室定位注射硫酸锌，分组如下：生理盐水对照组、30mM硫酸锌给药组，30mM硫酸锌给药+锌离子特异性螯合剂CQ腹腔注射组。在侧脑室注射6天后，麻醉取脑分离海马组织匀浆，用免疫印迹检测tau磷酸化水平的变化，以及PP2Ac Y307磷酸化水平的变化,检测PP2A的活性；细胞水平采用小鼠成神经瘤N2a细胞系，用100μM硫酸锌处理3小时后提取蛋白，同样方法检测PP2Ac Y307磷酸化及tau磷酸化水平。同时在细胞水平给予PP2或DES预孵育30min或转染野生型PP2A和突变性PP2A，在用用100μM硫酸锌处理3小时后提取蛋白，同样方法检测PP2Ac磷酸化及tau磷酸化水平。在转基因动物水平，我们采用转人tau的转基因小鼠，锌离子螯合剂CQ灌胃35天，检测PP2Ac磷酸化及tau磷酸化水平及可溶性tau和不溶性tau的变化情况。【结果】将锌离子直接注射入整体动物侧脑室，6天后海马PP2Ac Y307磷酸化水平明显增高，伴随PP2A活性下降，tau蛋白在多个丝氨酸/苏氨酸位点如Ser214、Thr205、Ser396、Ser404发生过度磷酸化；给动物同时腹腔注射锌离子螯合剂CQ,则可部分逆转PP2Ac Y307磷酸化水平和PP2A活性的抑制。用硫酸锌处理小鼠成神经瘤N2a细胞，同样导致PP2Ac发生酪氨酸磷酸化修饰和活性下降，tau蛋白在多个位点发生过度磷酸化,伴随Src失活性磷酸化位点酪氨酸529（Tyr529）磷酸化水平降低，Src激活。给予锌同时给予PP2A的激动剂或过表达野生型PP2A可逆转锌导致的PP2Ac活性下降以及tau蛋白的过度磷酸化。给予Src家族的特异性抑制剂PP2同样可部分逆转PP2Ac Y307磷酸化和PP2A活性的抑制以及tau蛋白的磷酸化。转基因小鼠的给予锌离子螯合剂CQ灌胃后，Src活性抑制，PP2Ac Y307磷酸化水平降低，PP2A活性抑制和tau蛋白磷酸化被逆转，伴随海马内不可溶性tau水平明显下降，【结论】锌离子可通过激活Src而对PP2A进行酪氨酸磷酸化修饰下调PP2A活性,从而导致tau蛋白发生过度磷酸化，促进AD样病变的形成。干预tau转基因小鼠脑内锌离子水平可上调PP2A活性并降低tau蛋白的磷酸化水平。因此干预锌离子水平有可能成为在体上调PP2A活性的新策略。第二部分锌离子体外直接抑制PP2A活性及机制研究我们已经在第一部分阐明锌离子在整体动物脑和细胞内可通过激活Src而使PP2AY307磷酸化失活和tau蛋白过度磷酸化。那么在体外锌离子对PP2A是否有直接调节作用？其内在机制是什么到目前为止尚无报道。【目的】阐明锌离子直接调节PP2A活性及其内在机制。【材料和方法】收集培养的N2a细胞并裂解，在不加入ATP的情况下直接将细胞裂解液和100μM硫酸锌或10nM冈田酸（OA）孵育0，5，15，30min，用免疫印迹检测tau磷酸化水平的变化。同样的方法用不同浓度（0，10，50，100μM）硫酸锌与细胞裂解液共孵育不同时间（0，5，15，30，60min），检测PP2A活性。构建不同片段的PP2Ac原核表达质粒并纯化，将纯化的GST-PP2A_(1-50)、PP2A_(51-270)和PP2A_(271-309)分别和100μM硫酸锌孵育，检测PP2A活性。最后我们用亲和层析柱（IDA）预先孵育硫酸锌或不孵育硫酸锌，分别让纯化的GST-PP2A_(1-50)、GST-PP2A_(51-270)和GST-PP2A_(271-309)自然流过IDA柱，然后用EDTA洗脱，收集不同组分的液体用免疫印迹检测GST的变化以确定不同片段PP2Ac与Zn的结合情况。【结果】将硫酸锌和细胞裂解液共孵育可延缓tau蛋白发生去磷酸化的进程，共孵育15min时已和强PP2A抑制剂冈田酸具有同等的抑制tau蛋白去磷酸化的效果，同时随着时间和硫酸锌浓度的变化，PP2A的活性被抑制，在10μM，30min时到达最低水平，抑制率下降30%。而且给予硫酸锌10μM时，PP2A的抑制效应有时间依赖性。说明在体外锌离子直接抑制PP2A活性。为了进一步确定锌离子对PP2A的直接抑制作用，将截断的PP2A与锌离子直接孵育发现锌离子可以直接抑制GST-PP2A_(51-270)片段的活性。通过IDA实验发现，，锌离子仅和GST-PP2A_(51-270)有直接结合作用。【结论】在体外，锌离子通过与PP2A_(51-270)结合发挥直接抑制PP2A活性的作用，这为阐明PP2A活性调节的全新途径提供了重要线索，并为AD的防治提供新靶点。
[Abstract]:Alzheimer 's Disease (AD) is the most common Alzheimer's disease. Its main pathological changes are the formation of neuronal fibrous tangles (NFTs) and senile plaques (SP) in the brain, the main component of the formation of.NFTs is the over phosphorylated microtubule related protein tau, and the abnormal phosphorylated tau loses its microtubule assembly and the function of stabilizing microtubules. The aggregation of PHF, which eventually leads to the formation of NFT, the degeneration of synapses and the loss of neurons, plays a key role in the development of AD, and the phosphorylation of tau is regulated mainly by related protein kinase (PK) and protein phosphorylase (protein phosphatases, PP). The activity of the esterase 2A (PP2A) decreases in the brain of Alzheimer's patients and participates in the formation of abnormal phosphorylation of AD like tau protein, but the upstream factors and mechanisms that lead to the decline of PP2A activity have not been elucidated.
Zinc (Zn) ion, one of the more abundant trace elements in the brain, participates in nerve conduction, antioxidant reaction, and regulates the activity of various enzymes and growth factors. A large number of studies have shown that the increase of the level of extracellular zinc ions can lead to tyrosine phosphorylation of a variety of proteins. The results of previous studies show that PP2A catalyzes the subunit caseine. After phosphorylation of the acid 307 site (Y307), the activity of PP2A can be inhibited obviously, but the level of zinc ion in the susceptible brain region of AD is increased, suggesting that zinc ions may participate in the down regulation of PP2A activity in the brain of AD and promote the excessive phosphorylation of tau protein. So far, the regulation and mechanism of Zn on PP2A have not yet been reported.
In this study, we focused on the regulation of zinc ions on PP2A and its role in tau overphosphorylation, and explained the direct and indirect inhibition mechanism of Zn on PP2A at the molecular level. The main results are as follows:
Part one
Zinc ions inhibit PP2A activity and lead to excessive phosphorylation of tau protein by activating Src pathway.
The activity of protein phosphatase 2A (PP2A) decreases in the human brain of Alzheimer's disease (AD) and participates in the formation of abnormal phosphorylation of AD like tau protein, but the upstream factors and mechanisms that lead to the decrease of PP2A activity have not been elucidated. It is reported that the phosphorylation of the tyrosine 307 points (Y307) of the PP2A catalyzed subunit is the direct cause of the deactivation of PP2A. However, it is not clear what mechanism makes PP2A inactive modification. A large number of results show that the increase of extracellular zinc ions can lead to tyrosine phosphorylation of various proteins, but the level of zinc ions in the susceptible brain regions of AD is higher, suggesting that zinc ions may participate in the down regulation of PP2A activity in the brain of AD and promote the excessive phosphorylation of tau protein.
[Objective] to elucidate whether zinc ion promotes abnormal abnormal phosphorylation of tau protein and regulates PP2A activity through regulating PP2A activity.
The experiment was carried out on the whole and the cell level. The overall level was used in the male SD rats and the lateral ventricle was injected with zinc sulfate. The groups were as follows: the saline control group, the 30mM zinc sulfate administration group, the zinc sulfate administration and the zinc ion specific chelating agent CQ intraperitoneal injection group. 6 days after the lateral ventricle injection, the brain was anesthetized to separate the hippocampus. Tissue homogenate was used to detect the changes of phosphorylation level of tau and the changes of PP2Ac Y307 phosphorylation level, and the activity of PP2A was detected. The cell level was used in the mouse neuroma N2a cell line, and the protein was extracted with 100 u M zinc sulfate for 3 hours. The same method was used to detect the phosphorylation of PP2Ac Y307 and the level of tau phosphorylation. PP2 or DES was preincubated with PP2 or DES to transfect wild type PP2A and mutant PP2A. The protein was extracted after 3 hours of treatment with 100 mu zinc sulfate. The same method was used to detect the phosphorylation of PP2Ac and the level of tau phosphorylation. At the level of transgenic animals, we used transgenic mice in transgenic mice and zinc ion chelating agent CQ for 35 days to detect PP2Ac phosphorylation and t. The level of Au phosphorylation and the change of soluble tau and insoluble tau.
[results] the zinc ions were injected directly into the whole animal lateral ventricle. After 6 days, the phosphorylation level of PP2Ac Y307 in the hippocampus was significantly increased, with the decrease of PP2A activity. The tau protein was over phosphorylated at several serine / threonine sites such as Ser214, Thr205, Ser396, Ser404, and the zinc ion chelating agent CQ was injected into the animals at the same time, then the P could be partly reversed P. P2Ac Y307 phosphorylation level and inhibition of PP2A activity. The treatment of mouse neuroma N2a cells with zinc sulfate also leads to tyrosine phosphorylation and activity degradation of PP2Ac, tau protein is over phosphorylated at multiple sites, with the decrease of tyrosine 529 (Tyr529) phosphorylation level and Src activation with the Src deactivation phosphorylation site. PP2A agonists or overexpressed wild type PP2A can reverse zinc induced PP2Ac activity and tau protein overphosphorylation. The specific inhibitor PP2 of the Src family can also partially reverse the phosphorylation of PP2Ac Y307 and the inhibition of PP2A activity and the phosphorylation of tau proteins. After the stomach, the activity of Src was inhibited, the level of phosphorylation of PP2Ac Y307 decreased, the activity of PP2A was inhibited and the phosphorylation of tau protein was reversed, and the level of insoluble tau in the hippocampus decreased significantly.
[Conclusion] zinc ions can reduce PP2A activity by tyrosine phosphorylation modification of PP2A by activating Src, resulting in excessive phosphorylation of tau protein and promoting the formation of AD like lesions. The intervention of zinc ion levels in the brain of tau transgenic mice can up regulate the activity of PP2A and reduce the phosphorylation level of tau protein. Therefore, the level of zinc ion intervention can be interfered with. It may be a new strategy for up regulation of PP2A activity in body.
The second part
Direct inhibition of PP2A activity by zinc ion and its mechanism
We have stated in the first part that zinc ions can be activated by Src in the whole animal brain and cells to deactivate the phosphorylation of PP2AY307 and to overphosphorylation of tau protein.
[Objective] to elucidate the direct regulation of zinc ion on PP2A activity and its underlying mechanism.
[materials and methods] the cultured N2a cells were collected and lysed. The cell lysates and 100 M zinc sulfate or 10nM Okada acid (OA) were incubated for 0,5,15,30min without ATP, and the changes in the phosphorylation level of tau were detected by immunoblotting. The same method was incubated with different concentrations (0,10,50100 u M) of zinc sulfate and cell lysate. At the same time (0,5,15,30,60min), the activity of PP2A was detected. The PP2Ac prokaryotic expression plasmid with different fragments was constructed and purified. The purified GST-PP2A_ (1-50), PP2A_ (51-270) and PP2A_ (271-309) were incubated with 100 mu zinc sulfate respectively, and the activity of PP2A was detected. Finally, we incubated zinc sulfate or no incubating zinc sulfate with the affinity chromatography column (IDA). GST-PP2A_ (1-50), GST-PP2A_ (51-270) and GST-PP2A_ (271-309) naturally flow through the IDA column, then EDTA eluted with EDTA, and the changes of GST in different fractions were detected by immunoblotting to determine the combination of PP2Ac and Zn in different segments.
[results] the incubation of zinc sulfate and cell lysate can delay the process of dephosphorylation of tau protein. When incubating for 15min, it has the same effect as the strong PP2A inhibitor, okadaic acid, which inhibits the dephosphorylation of tau protein. At the same time, the activity of PP2A is inhibited with the change of time and zinc sulfate concentration, and the lowest water is reached at 10 u M and 30min. The inhibition rate decreased by 30%. and when zinc sulfate was given to 10 mu M, the inhibitory effect of PP2A was time dependent. It indicated that zinc ions in vitro inhibited the activity of PP2A directly. In order to further determine the direct inhibitory effect of zinc ions on PP2A, the truncated PP2A and zinc ions were directly incubated and found that zinc ions could directly inhibit the activity of GST-PP2A_ (51-270) fragments. IDA experiments showed that zinc ions only had direct interaction with GST-PP2A_ (51-270).
[Conclusion] in vitro, zinc ions play a direct role in inhibiting PP2A activity by combining with PP2A_ (51-270). This provides an important clue to elucidate the new pathway of the regulation of PP2A activity and provide new targets for the prevention and control of AD.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】