SUMO修饰对Tau蛋白磷酸化及其降解的影响

发布时间：2018-06-09 03:19

本文选题：SUMO-1 + Tau　；参考：《华中科技大学》2012年博士论文

【摘要】：第一部分SUMO-1可诱导Tau蛋白过度磷酸化阿尔兹海默病(Alzheimer's disease, AD)是一种较为常见的、呈进行性发展的可致死性神经退行性疾病,其主要病理改变是以细胞内的神经原纤维缠结(neurofibrillary tangle, NFTs)和细胞外的老年斑(senile plaques, SPs)形成为特征。Tau蛋白是一种重要的微管相关蛋白,与微管一起构成神经细胞骨架结构,主要生理功能可促进微管蛋白聚合形成微管,维持其功能稳定。当Tau蛋白发生过度磷酸化时,将失去稳定微管的作用,并以其为中心导致NFTs的形成进而导致神经退行性病变。SUMO (small ubiquitin-like modifer)为小的泛素样修饰分子,含SUMO-1、SUMO-2、SUMO-3和SUMO-4四种类型,其功能是通过对底物蛋白进行SUMO修饰使底物蛋白的转录调控、底物蛋白功能和其在亚细胞器定位以及底物蛋白稳定性发生改变。底物蛋白的SUMO修饰可调节底物与其他蛋白之间的结合,即可干扰底物蛋白同其他蛋白的结合,又可为其他蛋白的结合提供位点,还可导致底物蛋白的空间构象发生改变从而暴露或隐藏了同其他蛋白的结合位点。SUMO与泛素可能存在相同的靶蛋白结合位点-赖氨酸残基,故有些靶蛋白SUMO化和其泛素化具有拮抗作用。现有研究已证明Tau能被SUMO-1修饰；导致Tau蛋白过度磷酸化的磷酸酯酶PP2A活性降低时可促进Tau的SUMO化；在AD转基因鼠中SUMO-1又能与磷酸化Tau共定位。由此提示Tau的磷酸化及其SUMO化二者之间具有某种特定的相关性。目的：通过过表达SUMO-1然后检测Tau的过度磷酸化水平,检测Tau蛋白SUMO化修饰对其过度磷酸化的影响；通过上调GSK-3p活性和下调PP2A活性引起Tau的过度磷酸化,检测Tau的磷酸化对其SUMO化修饰的调节,阐明Tau蛋白SUMO化和其磷酸化的相互关系。方法：在细胞水平,在稳定表达HEK293/Tau细胞中转染SUMO-1和在HEK293WT细胞中共同瞬时转染Tau+SUMO-1和共同瞬时转染突变TauK(340)R+SUMO-1使之过表达48小时,运用免疫印迹技术探讨SUMO-1过表达后Tau的磷酸化变化情况；在过表达SUMO-1基础上用PP2A抑制剂OA处理,观察Tau的磷酸化和其SUMO化变化；用SUMO特异性抑制剂Ginkgolic acid(GA)在细胞水平抑制SUMO-1再检测Tau蛋白磷酸化的变化情况。同时运用免疫荧光技术在细胞形态方面观察过表达SUMO-1时对Tau磷酸化的影响。在细胞和动物水平,上调GSK-3β活性后检测SUMO-1的表达水平变化情况及对Tau的SUMO化修饰影响。用免疫荧光技术观察人脑AD脑片中磷酸化Tau和SUMO-1的表达分布及共定位情况。结果：在本实验中我们观察到如下结果：(1)无论是瞬时转染Tau还是稳定表达Tau,过表达SUMO-1都可增加Tau的磷酸化水平；而共转染突变质粒Tau K340R和SUMO-1后Tau的磷酸化水平则没有明显变化。抑制SUMO-1可下调Tau的磷酸化水平。(2)无论是激活GSK-3p活性或抑制PP2Ac活性引起的Tau蛋白磷酸化都可增加Tau的SUMO化。在细胞内,SUMO-1可增加Tau的磷酸化并与其共定位。首次在人AD患者脑片内发现聚积的磷酸化Tau和SUMO-1共定位。结论：过表达SUMO-1可促进Tau的磷酸化；Tau的磷酸化也可增加其SUMO化。表明Tau的SUMO化与其磷酸化相互促进。第二部分SUMO-1通过抑制Tau的泛素化从而减缓Tau的降解,增加Tau的聚积导致AD的神经原纤维缠结(neurofibrillary tangle, NFTs)的主要成分是异常过度磷酸化的Tau蛋白。Tau蛋白属于微管相关蛋白,其正常生理功能是促进微管蛋白聚合形成微管并维持微管的功能稳定。当Tau异常过度磷酸化时,将失去稳定微管的作用,并以其为中心导致NFTs的形成并进而参与多种神经退行性疾病的发生发展。异常蛋白的降解主要由两大降解系统完成,即泛素-蛋白酶体降解途径(Ubiquitin-proteasome system, UPS)、溶酶体-自噬系统(autophagy-lysosome pathway)途径及内质网相关降解通路(endoplasmic retieulum-associated degradation pathway)。大多数细胞内的异常蛋白或长寿命蛋白主要是通过UPS降解。过度磷酸化的Tau蛋白也可通过UPS降解,但在AD患者中NFTs虽然能被泛素修饰,但却不断沉积,其原因尚不清楚。SUMO-1为小泛素样修饰分子,其功能是通过对底物蛋白进行SUMO化修饰可改变底物蛋白质的稳定性、蛋白质功能、底物蛋白在亚细胞器定位及转录调控。SUMO-1与泛素可能存在相同的靶蛋白结合位点-赖氨酸残基,靶蛋白SUMO化和泛素化可能具有拮抗作用,同时,靶蛋白SUMO化还可导致其空间结构发生改变,从而影响其通过UPS进行降解。现有研究证实Tau蛋白既能被泛素修饰,又能被SUMO-1修饰,抑制蛋白酶小体时Tau的SUMO化降低；Tau的磷酸化还可增加Tau的SUMO化。由此提示Tau的SUMO化和泛素化之间具有某种特定相关性。同时Tau在SUMO化修饰后是否影响其聚积尚不得而知。目的：探讨SUMO-1对Tau的降解及聚积过程的影响及其机制。方法：在细胞水平,在稳定表达HEK293/Tau细胞系中先转染SUMO-1质粒,然后用cycloheximide(GFX)处理进行蛋白质降解实验,利用免疫印迹技术检测Tau的降解情况及其机制。同样,在HEK293/wt细胞系中共瞬时转染Tau和SUMO-1质粒,再用GFX处理检测Tau的降解情况。同时在上述过表达Tau、突变Tau K340R和SUMO-1时提取可溶性与不可溶性Tau,检测Tau的聚积情况。结果：(1)过表达SUMO-1时Tau的降解受到抑制,当Tau蛋白第340位点由赖氨酸突变为精氨酸时,过表达SUMO-1后其降解有所增加。(2)无论是在293WT还是在293/Tau细胞中,过表达SUMO-1时Tau的泛素化均受到抑制。(3)过表达SUMO-1时,不可溶性Tau表达增多,而Tau的SUMO化位点突变后不可溶性Tau表达下降。结论：Tau在过表达SUMO-1时通过抑制泛素的表达,减少tau的多聚泛素化,可能进而阻遏泛素化的NFTs或异常过度磷酸化的Tau被蛋白酶小体识别,抑制其降解。Tau的SUMO化还可增加其聚积。
[Abstract]:Part SUMO-1 can induce excessive phosphorylation of Tau protein.
Alzheimer's disease (AD) is a more common and progressive neurodegenerative neurodegenerative disease. Its main pathological changes are the formation of.Tau protein characterized by neurofibrillary tangle (NFTs) and extracellular matrix (senile plaques, SPs) as a characteristic.Tau protein. Microtubule related proteins, together with microtubules, constitute the skeleton structure of the nerve cells. The main physiological functions can promote microtubule polymerization to form microtubules and maintain their function stability. When the Tau protein is overphosphorylated, it will lose the role of stable microtubule and lead to the formation of NFTs and lead to the neurodegenerative disease.SUMO (small Ubiquitin-like modifer) is a small ubiquitin modifier, containing four types of SUMO-1, SUMO-2, SUMO-3 and SUMO-4. The function is to regulate the transcriptional regulation of the substrate protein by SUMO modification to the substrate protein, the function of the substrate protein and the change in the localization of the subcellular organelles and the stability of the substrate protein. The SUMO modification of the substrate protein can be adjusted. The binding between the substrate and other proteins interferes with the binding of the substrate protein to other proteins, provides sites for the binding of other proteins, and can also lead to a change in the spatial conformation of the substrate protein, thus exposing or hiding the binding site.SUMO of the same protein with other proteins, which may have the same target protein binding site, lysine, and the ubiquitin. Acid residues, so some target protein SUMO and its ubiquitination have antagonism. Existing studies have shown that Tau can be modified by SUMO-1, and the phosphorylase PP2A activity of excessive phosphorylation of Tau protein can promote Tau SUMO; SUMO-1 in AD transgenic mice can also co localize with phosphorylated Tau. Thus, Tau phosphorylation and SUMO are suggested. There is a certain correlation between the two.
Objective: to detect the overphosphorylation of Tau by overexpression of SUMO-1 and to detect the effect of SUMO modification of Tau protein on its overphosphorylation, and to regulate the SUMO modification of Tau by up regulation of GSK-3p activity and down-regulation of PP2A activity, and to elucidate the interaction between SUMO and phosphorylation of Tau protein. Relationship.
Methods: at the cell level, transfection of SUMO-1 in stable expression of HEK293/Tau cells and the simultaneous transient transfection of Tau+SUMO-1 in HEK293WT cells and TauK (340) R+SUMO-1 of common transient transfection for 48 hours, the phosphorylation of Tau after SUMO-1 overexpression was studied by immunoblotting, and P was used to express SUMO-1 on the basis of P. P2A inhibitor OA was used to observe the phosphorylation and its SUMO changes of Tau; the changes in the phosphorylation of Tau protein were detected by the SUMO specific inhibitor Ginkgolic acid (GA) at the cell level, and the effect of SUMO-1 on the Tau phosphorylation was observed by immunofluorescence in cell morphology. In cell and animal water GSK-3 beta activity was up-regulated and the expression level of SUMO-1 was detected and the effect on the SUMO modification of Tau. The distribution and co localization of phosphorylated Tau and SUMO-1 in AD brain slices of human brain were observed by immunofluorescence.
Results: in this experiment, we observed the following results: (1) either transient transfection of Tau or stable expression of Tau, overexpression of SUMO-1 can increase the phosphorylation level of Tau, while the phosphorylation level of Tau in CO transfected plasmid Tau K340R and SUMO-1 can not be significantly changed. (2) no matter the level of phosphorylation of Tau. () The phosphorylation of Tau protein caused by activation of GSK-3p activity or inhibition of PP2Ac activity can increase the SUMO of Tau. In the cell, SUMO-1 can increase the phosphorylation of Tau and its co localization. The accumulation of Tau and SUMO-1 is found in the brain slices of human AD patients for the first time.
Conclusion: overexpression of SUMO-1 can promote the phosphorylation of Tau, and the phosphorylation of Tau can also increase its SUMO. It indicates that the SUMO of Tau and its phosphorylation promote each other. The second part of SUMO-1 reduces the degradation of Tau by inhibiting the ubiquitination of Tau and increases the accumulation of Tau.
The main component of neurofibrillary tangle (NFTs) that causes AD is abnormal and overphosphorylated Tau protein.Tau protein belongs to microtubule related protein. Its normal physiological function is to promote microtubule polymerization to form microtubules and maintain microtubule function stability. When Tau abnormal phosphorylation, the stable microtubule will be lost. Use and focus on the formation of NFTs and participate in the development of a variety of neurodegenerative diseases. The degradation of abnormal proteins is mainly done by two major degradation systems, that is, the ubiquitin proteasome degradation pathway (Ubiquitin-proteasome system, UPS), lysosome autophagy (autophagy-lysosome pathway) pathway and endoplasmic reticulum related The degradation pathway (endoplasmic retieulum-associated degradation pathway). Most of the abnormal proteins or long life proteins in the cells are degraded mainly through UPS. The excessive phosphorylation of Tau protein can also be degraded through UPS, but in AD patients, NFTs can be modified by ubiquitin, but it is not broken. The reason why.SUMO-1 is not clear is that.SUMO-1 is small ubiquitin. The function of the modifier is to alter the stability of the substrate protein by SUMO modification of the substrate protein, the function of protein, the protein function of the substrate and the regulation of.SUMO-1 and ubiquitin, which may have the same target protein binding site lysine residues, and the target protein SUMO and ubiquitination may have antagonistic effects. At the same time, the SUMO of the target protein can also cause its spatial structure to change, which affects its degradation through the UPS. The existing research confirms that the Tau protein can be modified by ubiquitin, and can be modified by SUMO-1, and the SUMO reduction of Tau is reduced when the protease body is suppressed. The phosphorylation of Tau can also increase the SUMO of Tau. Thus, the SUMO and ubiquitination of Tau can be suggested. There is a certain correlation between them. It is not known whether Tau will affect the accumulation of SUMO after modification.
Objective: To investigate the effect of SUMO-1 on the degradation and accumulation of Tau and its mechanism.
Methods: at the cell level, the SUMO-1 plasmid was transfected first in the stable expression HEK293/Tau cell line, then the protein degradation experiment was carried out with cycloheximide (GFX), and the degradation and mechanism of Tau were detected by immunoblotting technology. The Tau and SUMO-1 plasmids were transfected in the HEK293/wt cell line, and Tau was detected by GFX treatment. At the same time, soluble and insoluble Tau were extracted from above overexpressed Tau, mutant Tau K340R and SUMO-1, and the accumulation of Tau was detected.
Results: (1) the degradation of Tau was inhibited when SUMO-1 was overexpressed. When the 340th site of Tau protein was mutated from lysine to arginine, the degradation of SUMO-1 increased after overexpression. (2) the ubiquitination of Tau was inhibited when SUMO-1 was expressed in 293WT or 293/Tau cells. (3) the expression of insoluble Tau increased when SUMO-1 was overexpressed. However, the expression of insoluble Tau decreased after Tau SUMO mutation.
Conclusion: Tau can inhibit the polyubiquitination of tau by inhibiting the expression of ubiquitin and reducing the polyubiquitination of tau, which may further inhibit the ubiquitination of NFTs or abnormal overphosphorylation of Tau by the protease body recognition, and the inhibition of the SUMO degradation of the degradation of.Tau can also increase its accumulation.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

【共引文献】