【作者】 王秋艳；

【导师】 胡林森；

【作者基本信息】 吉林大学 ， 神经病学， 2007， 博士

【摘要】 目的研究PSI处理后PC12细胞的硝化、磷酸化和糖基化亚蛋白质组的变化,筛选出新的与PD、UPS相关的被异常修饰的蛋白,为PD的病因病理和临床治疗研究提供新的思路。方法建立PSI诱导的PC12细胞PD模型,提取细胞总蛋白,分别采用1)双向电泳、Western blot与质谱相结合的硝化亚蛋白质组学技术,2)双向电泳、Pro-Q Diamand磷酸化蛋白凝胶染色与质谱相结合的磷酸化亚蛋白质组学技术,3)双向电泳、Pro-Q Emerald 488糖基化蛋白凝胶染色与质谱相结合的糖基化亚蛋白质组学技术,获得差异蛋白点,并鉴定出差异蛋白。结果PSI处理后PC12细胞凋亡百分比为10%,大约在80%的细胞中诱导出包涵体;12个蛋白点的相对硝化水平发生显著改变,CCT 6a、lamin A、p60 protein和G3BP显著升高;24个蛋白点Pro-Q Diamand染色强度发生显著改变,分别为上调的TH、peripherin、p47和HSP27,以及下调的NPM和AR,同时TH、peripherin和HSP27的相对磷酸化水平显著升高,p47、NPM和AR的相对磷酸化水平显著下降;12个蛋白点Pro-Q Emerald 488染色强度发生显著改变,分别为上调的ORP150和annexin II,以及下调的p-ALDH,ORP150的相对糖基化水平显著升高,p-ALDH的相对糖基化水平显著下降,annexin II的相对糖基化水平无变化。讨论本研究成功地建立了蛋白酶体抑制的PD细胞模型,比较蛋白的硝化、磷酸化和糖基化水平的变化,分别探讨差异蛋白及其相关异常修饰在PD及PSI诱导的PC12细胞PD模型中可能的作用。本研究发现的CCT 6a、p60、lamin A、G3BP蛋白及其硝化修饰,p47、AR和NPM蛋白及其磷酸化修饰,annexin II蛋白及其糖基化修饰在以往关于PD的研究中均未被报道过,本研究也首次发现磷酸化的TH和HSP27、糖基化的ORP150和p-ALDH在蛋白酶体受到抑制后发生显著变化。这些发现为进一步阐明PD的发病机制和寻求PD药物治疗靶点提供了新的线索。更多还原

【Abstract】 Parkinson disease (PD) is a slowly progressive age-related neurodegenerative disorder that can be familial or sporadic, characterized with the preferential degeneration and loss of dopaminergic neuron of the substantia nigra pars compacta and the presence of Lewy body in the survival neurons. The etiology and etiopathogenesis of PD are still obscure, recent studies suggest that defects in the capacity of the ubiquitin-proteasome system (UPS) to degrade unwanted proteins may be a common feature in both of familial and sporadic forms of PD. The cell and animal models induced by proteasome inhibitors have been successfully established, which could be the closest model of PD so far.Nitration, phosphorylation and glycosylation of proteins are the important post-translation modifications (PTMs), and recent studies show that the abnormal modifications may play a key role in the etiopathogenesis and pathological process of PD. At present the researches of damages caused by abnormal PTMs in PD are carried out at single protein level, which depend on the precise hypothesis of proteins assumed to be modified and the attainment of antibody of the protein. The newly developed proteomic techniques of PTMs make it possible to find a batch of proteins that are modified in one experiment, so many abnormal modifications of proteins will be found, which can’t be or haven’t been discovered with the traditional methods.The aim of this study was to establish the PD model of PC12 cell with PSI, examine entirely the nitrated, phosphorylated and glycosylated proteins with the proteomic techniques of PTMs, analyze the impact of PSI on protein modifications, and find some new abnormally modified proteins which are related to PD and UPS.The PC12 cells were cultured with routine methods, then respectively exposed to DMSO (in the control group) and PSI (in the experiment group,disolved in DMSO). After 24 hours PC12 cells were harvested and the total proteins were extracted. The differences of protein nitration between two groups were obtained by two-dimensional gel electrophoresis (2-DE), Western blot and Coomasie Blue stain. The differences of protein phosphorylation between two groups were obtained by 2-DE, Pro-Q Diamand phosphoprotein stain and Coomasie Blue stain. The differences of protein glycosylation between two groups were obtained by 2-DE, Pro-Q Emerald 488 glycoprotein stain and Coomasie Blue stain. Then certain proteins were identified by MALDI-TOF MS.After exposed to PSI for 24 hours, 10% of PC12 cells were dead and cellular inclusions beside the nucleus were observed in 80% of the survival cells. The nitration of 12 protein spots were significantly changed, 4 of which were successfully identified by MALDI-TOF MS. They were chaperonin subunit 6a (CCT 6a), lamin A, p60 protein, and Ras-GTPase-activating protein SH3-domain binding protein (G3BP), the nitrations of which were increased significantlly. CCT and p60 are molecular-chaperone and molecular-chaperone cofactor, which involve in the cellular protein-quality control; lamin A is an intermediate filaments protein, participating in the construction of cytoskeleton; G3BP is a protein with endogenous ribonulease activity and ATP/Mg2+ dependent DNA/RNA helicase activity which binds to the SH3 domain of Ras-GTPase activating protein.After exposed to PSI for 24 hours, the Pro-Q Diamand intensity of 24 proteins were significantly changed, 6 of which were identified by MALDI-TOF MS. The phosphoryaltion of tyrosine hydroxylase (TH), peripherin, p47, and heat shock protein 27 (HSP27) were increased, while the phosphorylations of nucleolar phosphoprotein B23 (NPM) and aldehyde reductase 1 (AR) were decreased. Then the relative phosphorylation of the above proteins were further analyzed, that of peripherin, HSP27 and TH were increased and p47, NPM and AR were decreased. TH is the rate-limiting enzyme of the biosynthesis of catecholamine and the biomarker of dopaminergic neurons, which catalyzes the conversion of L-tyrosine to DOPA; peripherin is a III-type intermediate filament protein which exclusively expresses in the neurons of peripheral nervous system (PNS), and in the neurons of CNS directly projecting to the periphery, the biological functions of which, especially in the brain, are still poorly understood; p47 is a cofactor of p97 (a AAA-ATPase), which binds to p97 and participates in the memberane fusion of Golgi and endoplasmic reticulum (ER), as well as the degradation of a small subset of VCP-dependent UPS substrates; HSP27 belongs to the small heat shock proteins family (sHSP), main functions of which are to facilitate the folding of proteins and inhibit the aggregation of abnormal proteins; NPM is a nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm during the cell cycle and has several interacting partners and diverse cellular functions; AR belongs to the aldo-keto reductase superfamily, and catalyzes the conversion of aldehyde to acetic acid using NAD(P)(H) as a cofactor, that may have roles in glucose metabolism, aldehyde metabolism, and electron transport.After exposed to PSI for 24 hours, the intensity of Pro-Q Emerald 488 stain of 12 proteins were significantly changed, 3 of which were identified by MALDI-TOF MS. The glycosylations of 150kDa oxgen-regulated protein precursor (ORP150) and annexin II were increased,while the glycosylation of mitochondrial aldehyde dehydrogenase precursor (p-ALDH) was decreased. Then the relative glycosylation of the above proteins were analyzed, that of ORP150 was increased, p-ALDH was decreased and annexin II was unchanged. ORP150 locates in ER and is an ER-associated glycoprotein with molecular-chaperone activity; annexin II is involved in the formation of Adherens Junctions, endocytosis, exocytosis, the maintenance of cytoskeleton and so on; p-ALDH locates in the mitochondria, and is involved in the oxidation of aldehydes derived from neurotransmitters and lipid peroxidation.After searching on PubMed, we found that the nitrations and relative nitration levels of CCT 6a, p60, lamin A, G3BP, the phosphorylation and relative phosphorylation levels of p47, AR, NPM, the glycosylation and relative glycosylation level of annexin II, were not reported in previous studies of PD. Also, we first confirmed the changes of phosphorylated TH, HSP27 and their phosphorylation levels, glycosylated ORP150, p-ALDH and their glycosylation levels in PD model of PC12 treated with PSI. All these findings may provide new clues on the etiopathogenesis of PD and the targets of drug treatments.更多还原