【作者】 陈冬；

【导师】 王光辉；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2010， 博士

【摘要】 帕金森病(Parkinson’s Disease, PD),是一种极为常见的神经退行性疾病,其发病率仅次于阿尔兹海默症(AD),在临床上表现为一系列运动障碍,如静止性震颤、肌肉僵直、姿势反射障碍以及运动迟缓等。帕金森病的发病率和年龄显著正相关,在65岁以上人群发病率约为1%,但是在85岁以上人群发病率激增至3%左右。帕金森病的主要病理特征有两点,一是中脑黑质多巴胺能神经元的选择性死亡,二是在存活的神经元中路易小体(Lewy body)和路易神经突的形成。帕金森病多为散发病例,仅有10-15%的帕金森病属于家族遗传型帕金森病。帕金森病的病因尚不明确,但是环境以及遗传因素被认为是主要诱因。最近,陆续有一些帕金森病致病基因被鉴定出和家族型帕金森病相关,这些基因的突变或者缺失会导致其编码的蛋白质功能异常从而诱导发病。常染色体隐性遗传的青少年型帕金森病(AR-JP)是一种最为常见的家族型帕金森病,其特点是发病年龄较早,通常在40岁前发病。此病是由于parkin基因的突变而导致的。在所有的早发家族遗传型帕金森病人里有接近50%的病人是由于parkin突变导致的；而且在所有的早发型的散发帕金森病病人中,有15%的病人同样是由于parkin突变导致的。Parkin,又名PARK2,它是一个泛素蛋白酶体系统中的泛素连接酶(E3),其主要功能是在泛素激活酶(E1)和泛素结合酶(E2)的帮助下,识别特定的蛋白底物,通过泛素-蛋白酶体促进该蛋白降解。所以,对于parkin特定底物的寻找一直是帕金森病研究中的一个重点。近来,parkin也被报道可以通过行使其E3连接酶功能对特定底物蛋白的进行泛素化修饰而调控底物蛋白的下游功能,parkin的这一新发现被认为可能是帕金森病病因之一细胞内的蛋白质降解途径主要有俩种：即上文所提的泛素-蛋白酶体(ubiquitin-proteasom system)途径和细胞自噬(autophagy)途径,他们各自的降解终端分别是蛋白酶体和溶酶体。如果细胞的自噬降解途径发生紊乱,就会诱发神经退行性疾病以及未成熟的神经元的死亡,最近的研究更是直接证明自噬在帕金森病的发病过程中起重要作用。有趣的是,帕金森病相关基因parkin被报道在线粒体损伤剂CCCP处理下可以诱发自噬以清除受损线粒体；并且在parkin敲除鼠模型中,已经发现了自噬的标志蛋白LC3-Ⅱ型蛋白的上调,这些都揭示了parkin与自噬存在联系。但是到目前为止,parkin的功能及其在帕金森病发病过程它与自噬的关系仍不清楚。Parkin及其发病相关突变体与帕金森病的发病过程以及自噬的关系是我们的研究重点。本文中,我们通过体外以及体内的蛋白结合实验发现,parkin可以特异性的通过其蛋白结构C端的RING1-IBR-RING2吉构与抗凋亡蛋白Bcl-2结合,而不与Bax或者Bcl-xl这俩个Bcl-2家族蛋白结合。进一步研究发现parkin可以通过行使其E3连接酶功能,单泛素化修饰Bcl-2来延长其半衰期,从而上调Bcl-2蛋白含量。所增加的Bcl-2可以结合更多自噬相关蛋白Beclin 1,形成稳定的复合体而抑制自噬,而parkin的发病相关的E3活性受损突变体则丧失了此功能。本研究提示了parkin在细胞自噬的调控过程中起了重要作用,为更进一步了解parkin的对细胞保护机制,以及帕金森病的分子病理机制提供了新思路。更多还原

【Abstract】 Parkinson’s disease (PD) is the second most common neurodegenerative disorders after Alzheimer’s disease and is characterized by a distinct set of motor symptoms, including tremor, muscle rigidity, postural instability and bradykinesia. Aging is the most risk factor in pathogenesis of PD which is estimated to affect approximately 1% of the population at 65 years and increased to 3% at 85. PD is characterized by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and by the presence of Lewy bodies (LBs) and Lewy neurites in living neurons. Most of PD patients appear to be sporadic and just approximately 10-15% of PD patients are heriatable PD. Although the cause of PD is poorly understood, there is evidence that both environmental factors and genetic factors contribute to its development. Recently, several genes have been reported to be associated with the pathogenesis of familial forms of PD. Mutations or deletions of these genes leading to dysfunction of proteins are contributed to pathogenesis of PD.Autosomal recessive juvenile-onset parkinsonism (AR-JP) is a common familial forms of PD, and one notable distinct feature is early-onset before 40 years old. Mutations in the parkin gene have been reported to cause AR-JP. It has been shown that mutations in parkin account for nearly 50% of patients with the early-onset familial PD cases and more than 15% of sporadic PD cases with early onset. Parkin, also named PARK2, is an E3 ubiquitin ligase in ubiquitin-proteasome system (UPS). It works with El buiqui tin-activating enzyme and E2 ubiqui tin-conjugating enzyme to recognize specialy target proteins and enhances their degradation via UPS. So, searching for the new specific substrate of parkin is a hot spot in PD research. Recently, parkin has been reported to ubiquitinate particular protein substrate via its E3 ubiquitin ligase activity to change the substrate to degradation-independent role, and this may be important for molecular pathogenesis of parkin-linked PD.The ecukaryotic cells have two main protein degradation pathways:the ubiquitin-proteosome pathway and the autophagy-lysosome pathway. Previous studies have shown that the selective ablation of autophagy gene could directly cause neurodegenerative disease. An early report shows that autophagy is important for pathogenesis of PD. These results suggest that autophagy is associated with PD and neurodegeneration. A recent study indicates that in the neuronal cultures of midbrain from parkin null mice, markers of autophagy, such as LC3-Ⅱ/Ⅰ, are increased suggesting that knockout of parkin may induce autophagy. However, the links among parkin, autophagy and PD is still not clear. So, in this study, we focus on the relationship between parkin and autophagy.We report here that Bcl-2 is a novel substrate for the E3 ubiquitin ligase parkin. Parkin directly interactes with Bcl-2 specifically through its RING1-IBR-RING2 domain in C-terminus and mediates the mono-ubiquitination of Bcl-2. The mono-ubiquitination of Bcl-2 by parkin alters Bcl-2 turnover to increase its protein level. The increased Bcl-2 could bind more Beclin 1 to inhibit autophagy. The PD-linked mutant forms of parkin whose E3 ligase activity are selectively impaired, fail to regulate the mono-ubiquitination of Bcl-2.These results identify parkin as a novel regulator of autophagy through mono-ubiquitinating Bcl-2 and suggest a new way to understand the protect effects of parkin.更多还原