本文選題：鋅 + 蛋白磷酸酶��； 參考：《華中科技大學(xué)》2013年博士論文

【摘要】：阿爾茨海默�。ˋlzheimer’s Disease, AD）是最常見的老年性癡呆癥，其主要病理學(xué)改變是腦內(nèi)神經(jīng)元纖維纏結(jié)（NFTs）和老年斑(SP)的形成。NFTs的主要成分是過度磷酸化的微管相關(guān)蛋白tau,異常磷酸化的tau喪失其促微管組裝和穩(wěn)定微管的功能,并聚集成為成對螺旋絲（PHF），最終導(dǎo)致NFT的形成、突觸的退變和神經(jīng)元的丟失,在AD的發(fā)生、發(fā)展中起關(guān)鍵作用。而tau的磷酸化主要受相關(guān)蛋白激酶（protein kinases, PK）和蛋白磷酸酯酶（protein phosphatases,PP）的調(diào)節(jié)。大量的研究事實表明，蛋白磷酸酯酶2A(PP2A)的活性在阿爾茨海默病人腦中下降并參與AD樣tau蛋白異常磷酸化的形成，但導(dǎo)致PP2A活性下降的上游因素和機制尚未闡明。 鋅（Zn）離子，是腦內(nèi)含量較為豐富的微量元素之一，參與神經(jīng)傳導(dǎo)，抗氧化反應(yīng)，并調(diào)節(jié)多種酶和生長因子的活性。大量研究結(jié)果表明，胞外鋅離子水平增高可導(dǎo)致多種蛋白質(zhì)發(fā)生酪氨酸磷酸化修飾。前期研究結(jié)果顯示PP2A催化亞單位酪氨酸307位點（Y307）磷酸化后可明顯抑制PP2A活性，而在AD易感腦區(qū)鋅離子水平增高，提示鋅離子可能參與了AD腦內(nèi)PP2A活性的下調(diào)而促進tau蛋白過度磷酸化。且到目前為止Zn對PP2A的調(diào)節(jié)及機制尚無報道。 我們在本研究中重點探討了鋅離子對PP2A的調(diào)節(jié)及其在tau過度磷酸化中的作用，并且從分子水平解釋了Zn對PP2A的直接和間接抑制作用機制。主要結(jié)果如下： 第一部分 鋅離子通過激活Src途徑抑制PP2A活性和導(dǎo)致tau蛋白過度磷酸化 蛋白磷酸酯酶2A(PP2A)的活性在阿爾茨海默�。ˋD）人腦中下降并參與AD樣tau蛋白異常磷酸化的形成，但導(dǎo)致PP2A活性下降的上游因素和機制尚未闡明。有文獻報道顯示，PP2A催化亞基的酪氨酸307位點（Y307）磷酸化修飾是導(dǎo)致PP2A失活的直接原因，但何種機制促使PP2A發(fā)生失活性修飾還不清楚。大量研究結(jié)果表明，胞外鋅離子水平增高可導(dǎo)致多種蛋白質(zhì)發(fā)生酪氨酸磷酸化修飾，而在AD易感腦區(qū)鋅離子水平增高，提示鋅離子可能參與了AD腦內(nèi)PP2A活性的下調(diào)而促進tau蛋白過度磷酸化。 【目的】闡明鋅離子是否通過調(diào)節(jié)PP2A活性促進tau蛋白異常過度磷酸化及調(diào)節(jié)PP2A活性的機制。 【材料和方法】實驗在整體和細胞水平進行。整體水平采用雄性SD大鼠，側(cè)腦室定位注射硫酸鋅，分組如下：生理鹽水對照組、30mM硫酸鋅給藥組，30mM硫酸鋅給藥+鋅離子特異性螯合劑CQ腹腔注射組。在側(cè)腦室注射6天后，麻醉取腦分離海馬組織勻漿，用免疫印跡檢測tau磷酸化水平的變化，以及PP2Ac Y307磷酸化水平的變化,檢測PP2A的活性；細胞水平采用小鼠成神經(jīng)瘤N2a細胞系，用100μM硫酸鋅處理3小時后提取蛋白，同樣方法檢測PP2Ac Y307磷酸化及tau磷酸化水平。同時在細胞水平給予PP2或DES預(yù)孵育30min或轉(zhuǎn)染野生型PP2A和突變性PP2A，在用用100μM硫酸鋅處理3小時后提取蛋白，同樣方法檢測PP2Ac磷酸化及tau磷酸化水平。在轉(zhuǎn)基因動物水平，我們采用轉(zhuǎn)人tau的轉(zhuǎn)基因小鼠，鋅離子螯合劑CQ灌胃35天，檢測PP2Ac磷酸化及tau磷酸化水平及可溶性tau和不溶性tau的變化情況。 【結(jié)果】將鋅離子直接注射入整體動物側(cè)腦室，6天后海馬PP2Ac Y307磷酸化水平明顯增高，伴隨PP2A活性下降，tau蛋白在多個絲氨酸/蘇氨酸位點如Ser214、Thr205、Ser396、Ser404發(fā)生過度磷酸化；給動物同時腹腔注射鋅離子螯合劑CQ,則可部分逆轉(zhuǎn)PP2Ac Y307磷酸化水平和PP2A活性的抑制。用硫酸鋅處理小鼠成神經(jīng)瘤N2a細胞，同樣導(dǎo)致PP2Ac發(fā)生酪氨酸磷酸化修飾和活性下降，tau蛋白在多個位點發(fā)生過度磷酸化,伴隨Src失活性磷酸化位點酪氨酸529（Tyr529）磷酸化水平降低，Src激活。給予鋅同時給予PP2A的激動劑或過表達野生型PP2A可逆轉(zhuǎn)鋅導(dǎo)致的PP2Ac活性下降以及tau蛋白的過度磷酸化。給予Src家族的特異性抑制劑PP2同樣可部分逆轉(zhuǎn)PP2Ac Y307磷酸化和PP2A活性的抑制以及tau蛋白的磷酸化。轉(zhuǎn)基因小鼠的給予鋅離子螯合劑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蛋白磷酸化被逆轉(zhuǎn)，伴隨海馬內(nèi)不可溶性tau水平明顯下降， 【結(jié)論】鋅離子可通過激活Src而對PP2A進行酪氨酸磷酸化修飾下調(diào)PP2A活性,從而導(dǎo)致tau蛋白發(fā)生過度磷酸化，促進AD樣病變的形成。干預(yù)tau轉(zhuǎn)基因小鼠腦內(nèi)鋅離子水平可上調(diào)PP2A活性并降低tau蛋白的磷酸化水平。因此干預(yù)鋅離子水平有可能成為在體上調(diào)PP2A活性的新策略。 第二部分 鋅離子體外直接抑制PP2A活性及機制研究 我們已經(jīng)在第一部分闡明鋅離子在整體動物腦和細胞內(nèi)可通過激活Src而使PP2AY307磷酸化失活和tau蛋白過度磷酸化。那么在體外鋅離子對PP2A是否有直接調(diào)節(jié)作用？其內(nèi)在機制是什么到目前為止尚無報道。 【目的】闡明鋅離子直接調(diào)節(jié)PP2A活性及其內(nèi)在機制。 【材料和方法】收集培養(yǎng)的N2a細胞并裂解，在不加入ATP的情況下直接將細胞裂解液和100μM硫酸鋅或10nM岡田酸（OA）孵育0，5，15，30min，用免疫印跡檢測tau磷酸化水平的變化。同樣的方法用不同濃度（0，10，50，100μM）硫酸鋅與細胞裂解液共孵育不同時間（0，5，15，30，60min），檢測PP2A活性。構(gòu)建不同片段的PP2Ac原核表達質(zhì)粒并純化，將純化的GST-PP2A_(1-50)、PP2A_(51-270)和PP2A_(271-309)分別和100μM硫酸鋅孵育，檢測PP2A活性。最后我們用親和層析柱（IDA）預(yù)先孵育硫酸鋅或不孵育硫酸鋅，分別讓純化的GST-PP2A_(1-50)、GST-PP2A_(51-270)和GST-PP2A_(271-309)自然流過IDA柱，然后用EDTA洗脫，收集不同組分的液體用免疫印跡檢測GST的變化以確定不同片段PP2Ac與Zn的結(jié)合情況。 【結(jié)果】將硫酸鋅和細胞裂解液共孵育可延緩tau蛋白發(fā)生去磷酸化的進程，共孵育15min時已和強PP2A抑制劑岡田酸具有同等的抑制tau蛋白去磷酸化的效果，同時隨著時間和硫酸鋅濃度的變化，PP2A的活性被抑制，在10μM，30min時到達最低水平，抑制率下降30%。而且給予硫酸鋅10μM時，PP2A的抑制效應(yīng)有時間依賴性。說明在體外鋅離子直接抑制PP2A活性。為了進一步確定鋅離子對PP2A的直接抑制作用，將截斷的PP2A與鋅離子直接孵育發(fā)現(xiàn)鋅離子可以直接抑制GST-PP2A_(51-270)片段的活性。通過IDA實驗發(fā)現(xiàn)，，鋅離子僅和GST-PP2A_(51-270)有直接結(jié)合作用。 【結(jié)論】在體外，鋅離子通過與PP2A_(51-270)結(jié)合發(fā)揮直接抑制PP2A活性的作用，這為闡明PP2A活性調(diào)節(jié)的全新途徑提供了重要線索，并為AD的防治提供新靶點。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R749.16

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