本文選題：SUMO-1 + Tau��； 參考：《華中科技大學》2012年博士論文

【摘要】：第一部分SUMO-1可誘導Tau蛋白過度磷酸化 阿爾茲海默病(Alzheimer's disease, AD)是一種較為常見的、呈進行性發(fā)展的可致死性神經退行性疾病,其主要病理改變是以細胞內的神經原纖維纏結(neurofibrillary tangle, NFTs)和細胞外的老年斑(senile plaques, SPs)形成為特征。Tau蛋白是一種重要的微管相關蛋白,與微管一起構成神經細胞骨架結構,主要生理功能可促進微管蛋白聚合形成微管,維持其功能穩(wěn)定。當Tau蛋白發(fā)生過度磷酸化時,將失去穩(wěn)定微管的作用,并以其為中心導致NFTs的形成進而導致神經退行性病變。SUMO (small ubiquitin-like modifer)為小的泛素樣修飾分子,含SUMO-1、SUMO-2、SUMO-3和SUMO-4四種類型,其功能是通過對底物蛋白進行SUMO修飾使底物蛋白的轉錄調控、底物蛋白功能和其在亞細胞器定位以及底物蛋白穩(wěn)定性發(fā)生改變。底物蛋白的SUMO修飾可調節(jié)底物與其他蛋白之間的結合,即可干擾底物蛋白同其他蛋白的結合,又可為其他蛋白的結合提供位點,還可導致底物蛋白的空間構象發(fā)生改變從而暴露或隱藏了同其他蛋白的結合位點。SUMO與泛素可能存在相同的靶蛋白結合位點-賴氨酸殘基,故有些靶蛋白SUMO化和其泛素化具有拮抗作用�，F(xiàn)有研究已證明Tau能被SUMO-1修飾；導致Tau蛋白過度磷酸化的磷酸酯酶PP2A活性降低時可促進Tau的SUMO化；在AD轉基因鼠中SUMO-1又能與磷酸化Tau共定位。由此提示Tau的磷酸化及其SUMO化二者之間具有某種特定的相關性。 目的：通過過表達SUMO-1然后檢測Tau的過度磷酸化水平,檢測Tau蛋白SUMO化修飾對其過度磷酸化的影響；通過上調GSK-3p活性和下調PP2A活性引起Tau的過度磷酸化,檢測Tau的磷酸化對其SUMO化修飾的調節(jié),闡明Tau蛋白SUMO化和其磷酸化的相互關系。 方法：在細胞水平,在穩(wěn)定表達HEK293/Tau細胞中轉染SUMO-1和在HEK293WT細胞中共同瞬時轉染Tau+SUMO-1和共同瞬時轉染突變TauK(340)R+SUMO-1使之過表達48小時,運用免疫印跡技術探討SUMO-1過表達后Tau的磷酸化變化情況；在過表達SUMO-1基礎上用PP2A抑制劑OA處理,觀察Tau的磷酸化和其SUMO化變化；用SUMO特異性抑制劑Ginkgolic acid(GA)在細胞水平抑制SUMO-1再檢測Tau蛋白磷酸化的變化情況。同時運用免疫熒光技術在細胞形態(tài)方面觀察過表達SUMO-1時對Tau磷酸化的影響。在細胞和動物水平,上調GSK-3β活性后檢測SUMO-1的表達水平變化情況及對Tau的SUMO化修飾影響。用免疫熒光技術觀察人腦AD腦片中磷酸化Tau和SUMO-1的表達分布及共定位情況。 結果：在本實驗中我們觀察到如下結果：(1)無論是瞬時轉染Tau還是穩(wěn)定表達Tau,過表達SUMO-1都可增加Tau的磷酸化水平；而共轉染突變質粒Tau K340R和SUMO-1后Tau的磷酸化水平則沒有明顯變化。抑制SUMO-1可下調Tau的磷酸化水平。(2)無論是激活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。在細胞內,SUMO-1可增加Tau的磷酸化并與其共定位。首次在人AD患者腦片內發(fā)現(xiàn)聚積的磷酸化Tau和SUMO-1共定位。 結論：過表達SUMO-1可促進Tau的磷酸化；Tau的磷酸化也可增加其SUMO化。表明Tau的SUMO化與其磷酸化相互促進。第二部分SUMO-1通過抑制Tau的泛素化從而減緩Tau的降解,增加Tau的聚積 導致AD的神經原纖維纏結(neurofibrillary tangle, NFTs)的主要成分是異常過度磷酸化的Tau蛋白。Tau蛋白屬于微管相關蛋白,其正常生理功能是促進微管蛋白聚合形成微管并維持微管的功能穩(wěn)定。當Tau異常過度磷酸化時,將失去穩(wěn)定微管的作用,并以其為中心導致NFTs的形成并進而參與多種神經退行性疾病的發(fā)生發(fā)展。異常蛋白的降解主要由兩大降解系統(tǒng)完成,即泛素-蛋白酶體降解途徑(Ubiquitin-proteasome system, UPS)、溶酶體-自噬系統(tǒng)(autophagy-lysosome pathway)途徑及內質網相關降解通路(endoplasmic retieulum-associated degradation pathway)。大多數(shù)細胞內的異常蛋白或長壽命蛋白主要是通過UPS降解。過度磷酸化的Tau蛋白也可通過UPS降解,但在AD患者中NFTs雖然能被泛素修飾,但卻不斷沉積,其原因尚不清楚。SUMO-1為小泛素樣修飾分子,其功能是通過對底物蛋白進行SUMO化修飾可改變底物蛋白質的穩(wěn)定性、蛋白質功能、底物蛋白在亞細胞器定位及轉錄調控。SUMO-1與泛素可能存在相同的靶蛋白結合位點-賴氨酸殘基,靶蛋白SUMO化和泛素化可能具有拮抗作用,同時,靶蛋白SUMO化還可導致其空間結構發(fā)生改變,從而影響其通過UPS進行降解。現(xiàn)有研究證實Tau蛋白既能被泛素修飾,又能被SUMO-1修飾,抑制蛋白酶小體時Tau的SUMO化降低；Tau的磷酸化還可增加Tau的SUMO化。由此提示Tau的SUMO化和泛素化之間具有某種特定相關性。同時Tau在SUMO化修飾后是否影響其聚積尚不得而知。 目的：探討SUMO-1對Tau的降解及聚積過程的影響及其機制。 方法：在細胞水平,在穩(wěn)定表達HEK293/Tau細胞系中先轉染SUMO-1質粒,然后用cycloheximide(GFX)處理進行蛋白質降解實驗,利用免疫印跡技術檢測Tau的降解情況及其機制。同樣,在HEK293/wt細胞系中共瞬時轉染Tau和SUMO-1質粒,再用GFX處理檢測Tau的降解情況。同時在上述過表達Tau、突變Tau K340R和SUMO-1時提取可溶性與不可溶性Tau,檢測Tau的聚積情況。 結果：(1)過表達SUMO-1時Tau的降解受到抑制,當Tau蛋白第340位點由賴氨酸突變?yōu)榫彼釙r,過表達SUMO-1后其降解有所增加。(2)無論是在293WT還是在293/Tau細胞中,過表達SUMO-1時Tau的泛素化均受到抑制。(3)過表達SUMO-1時,不可溶性Tau表達增多,而Tau的SUMO化位點突變后不可溶性Tau表達下降。 結論：Tau在過表達SUMO-1時通過抑制泛素的表達,減少tau的多聚泛素化,可能進而阻遏泛素化的NFTs或異常過度磷酸化的Tau被蛋白酶小體識別,抑制其降解。Tau的SUMO化還可增加其聚積。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R749.16

【共引文獻】

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