Numb蛋白对α-synuclein蛋白的寡泛素化水平调控
发布时间：2018-10-18 12:54
【摘要】：背景 帕金森病（Parkinson’s disease, PD）是一种常见的神经退行性疾病，以运动迟缓，肌强直和静止性震颤为主要临床特征。在帕金森患者脑内，死亡的神经细胞多是位于炓质区的多巴胺神经元且多在临床症状之前出现，，当有明显临床症状时，大约有70%多巴胺神经元可能已经死亡。帕金森病的病理特征是多巴胺神经元中α-synuclein（α-syn）包涵体的出现，该蛋白是路易小体（Lewy body）的主要成分。异常折叠聚集的α-syn蛋白可形成低聚物，产生细胞毒性，是帕金森病致病的的主要病理过程。 Numb蛋白是一种进化保守的膜受体蛋白，在果蝇神经系统研究中发现其具有细胞命运决定子的作用。它具有多种蛋白质-蛋白质相互作用的区域，包括磷酸酪氨酸结合域（PTB）和羧基端结构域，PTB结构域可与泛素连接酶，如Siah1、Lnx等相互作用，对靶蛋白进行泛素化修饰。羧基端结构域包含一个EH结构域结合基序和α-衔接蛋白结合位点，可与相关靶蛋白结合，参与调控受体蛋白胞内循环、胞内移行。Numb是一种内吞蛋白，参与神经突生长和细胞迁移过程。此外，Numb通过与Notch相互拮抗，参与调节多种生化信号途径、调节粘附和紧密连接等诸多过程。有研究表明Numb/Notch信号通路通过调控泛素蛋白酶体系统，参与某些蛋白的泛素化调控。 关于Numb蛋白对α-syn蛋白寡泛素化水平的调控在国内外尚未见相关报道，本课题将深入探求Numb蛋白是否通过参与调控α-syn蛋白寡泛素化修饰模式，参与帕金森病的致病过程。为帕金森病的早期诊断与治疗提供新的思路和方法。 目的 探索Numb蛋白对α-突触核蛋白（α-synuclein，α-syn）寡泛素化水平的调控。 方法 将EGFP-N1、EGFP-Numb分别与HA-α-syn转染SH-SY5Y细胞；利用细胞免疫荧光法检测α-syn蛋白和Numb蛋白的亚细胞共定位；利用免疫共沉淀的方法检测Numb蛋白与α-syn蛋白的相互结合；利用Western blot技术检测Numb蛋白对α-syn蛋白寡泛素水平的调控；在MPP+细胞模型中利用细胞免疫荧光检测Numb对α-syn包涵体形成的影响。 结果 Numb与α-syn在细胞质中存在亚细胞共定位； Numb蛋白上调α-syn寡泛素化水平； Numb蛋白通过促使α-syn寡泛素水平上调诱导MPP+细胞模型中α-syn阳性包涵体形成。 结论 Numb蛋白上调α-syn蛋白寡泛素水平并促进α-syn包涵体形成。
[Abstract]:Background Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease characterized by motor retardation, myotonia and static tremor. In Parkinson's patients, most of the dead neurons are dopamine neurons located in the parkinsonian region and most of them appear before the clinical symptoms. When there are obvious clinical symptoms, about 70% of the dopamine neurons may have died. The pathological feature of Parkinson's disease is the presence of 伪-synuclein (伪-syn) inclusion bodies in dopamine neurons, which is the main component of Louie body (Lewy body). The abnormal folding and aggregation of 伪-syn protein can form oligomers and produce cytotoxicity, which is the main pathogenetic process of Parkinson's disease. Numb protein is an evolutionarily conserved membrane receptor protein. In the study of the nervous system of Drosophila melanogaster has been found to have the role of cell fate determinant. It has a variety of protein-protein interaction domains, including phosphotyrosine binding domain (PTB) and carboxyl terminal domain, and PTB domain can interact with ubiquitin ligases, such as Siah1,Lnx, to modify the target proteins. The carboxyl terminal domain contains a binding motif of EH domain and a binding site of 伪 -binding protein, which can bind to related target proteins and regulate the intracellular circulation of receptor proteins. Numb is an endocytosis protein. Participate in the process of neurite growth and cell migration. In addition, Numb interacts with Notch and participates in many processes, such as regulation of many biochemical signaling pathways, adhesion and tight junctions. Some studies have shown that the Numb/Notch signaling pathway participates in the regulation of ubiquitin by regulating the ubiquitin proteasome system. There have been no reports on the regulation of 伪-syn protein oligoubiquitization by Numb protein. This paper will explore whether Numb protein participates in the pathogenesis of Parkinson's disease by participating in the regulation of 伪-syn protein oligoubiquitin modification model. To provide new ideas and methods for early diagnosis and treatment of Parkinson's disease. Objective to investigate the regulation of 伪-synuclein, 伪-syn oligoubiquitin by Numb protein. Methods EGFP-N1,EGFP-Numb and HA- 伪 syn were transfected into SH-SY5Y cells, subcellular co-localization of 伪 syn protein and Numb protein was detected by immunofluorescence assay, and the interaction of Numb protein and 伪 syn protein was detected by immunoprecipitation. The effect of Numb on the formation of 伪 -syn inclusion bodies was detected by using Western blot technique, and the effect of Numb on the formation of 伪 -syn inclusion bodies in MPP cell model was detected by using Numb protein to regulate the level of 伪-syn protein oligoubiquitin. Results there were subcellular co-localization of Numb and 伪-syn in cytoplasm, Numb protein upregulated the level of 伪-syn oligobinylation. Numb protein induces the formation of 伪-syn positive inclusion bodies in MPP cells by up-regulating 伪-syn oligoubiquitin levels. Conclusion Numb protein upregulated the level of 伪-syn oligoubiquitin and promoted the formation of 伪-syn inclusion bodies.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
本文编号：2279185
[Abstract]:Background Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease characterized by motor retardation, myotonia and static tremor. In Parkinson's patients, most of the dead neurons are dopamine neurons located in the parkinsonian region and most of them appear before the clinical symptoms. When there are obvious clinical symptoms, about 70% of the dopamine neurons may have died. The pathological feature of Parkinson's disease is the presence of 伪-synuclein (伪-syn) inclusion bodies in dopamine neurons, which is the main component of Louie body (Lewy body). The abnormal folding and aggregation of 伪-syn protein can form oligomers and produce cytotoxicity, which is the main pathogenetic process of Parkinson's disease. Numb protein is an evolutionarily conserved membrane receptor protein. In the study of the nervous system of Drosophila melanogaster has been found to have the role of cell fate determinant. It has a variety of protein-protein interaction domains, including phosphotyrosine binding domain (PTB) and carboxyl terminal domain, and PTB domain can interact with ubiquitin ligases, such as Siah1,Lnx, to modify the target proteins. The carboxyl terminal domain contains a binding motif of EH domain and a binding site of 伪 -binding protein, which can bind to related target proteins and regulate the intracellular circulation of receptor proteins. Numb is an endocytosis protein. Participate in the process of neurite growth and cell migration. In addition, Numb interacts with Notch and participates in many processes, such as regulation of many biochemical signaling pathways, adhesion and tight junctions. Some studies have shown that the Numb/Notch signaling pathway participates in the regulation of ubiquitin by regulating the ubiquitin proteasome system. There have been no reports on the regulation of 伪-syn protein oligoubiquitization by Numb protein. This paper will explore whether Numb protein participates in the pathogenesis of Parkinson's disease by participating in the regulation of 伪-syn protein oligoubiquitin modification model. To provide new ideas and methods for early diagnosis and treatment of Parkinson's disease. Objective to investigate the regulation of 伪-synuclein, 伪-syn oligoubiquitin by Numb protein. Methods EGFP-N1,EGFP-Numb and HA- 伪 syn were transfected into SH-SY5Y cells, subcellular co-localization of 伪 syn protein and Numb protein was detected by immunofluorescence assay, and the interaction of Numb protein and 伪 syn protein was detected by immunoprecipitation. The effect of Numb on the formation of 伪 -syn inclusion bodies was detected by using Western blot technique, and the effect of Numb on the formation of 伪 -syn inclusion bodies in MPP cell model was detected by using Numb protein to regulate the level of 伪-syn protein oligoubiquitin. Results there were subcellular co-localization of Numb and 伪-syn in cytoplasm, Numb protein upregulated the level of 伪-syn oligobinylation. Numb protein induces the formation of 伪-syn positive inclusion bodies in MPP cells by up-regulating 伪-syn oligoubiquitin levels. Conclusion Numb protein upregulated the level of 伪-syn oligoubiquitin and promoted the formation of 伪-syn inclusion bodies.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5
【参考文献】
相关期刊论文 前1条
1 荆婧;王雪晶;马耀华;祝应俊;丁晓岚;滕军放;;α-synuclein(A53T)蛋白调控自噬诱导神经元凋亡[J];中国神经精神疾病杂志;2013年02期
本文编号：2279185
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