细胞氧化应激过程中受PRAK调控的磷酸化蛋白的鉴定
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摘要
氧自由基,也称活性氧簇(reactive oxygen species, ROS),包括超氧阴离子(·O2-)、羟自由基(·OH)、过氧化氢(H2O2)、一氧化氮(NO·)等,是细胞内多种正常代谢过程的产物并在生物体中扮演着双重角色。在活性氧簇处于低浓度或者适当浓度时,它对细胞免疫应答非常关键,如在对抗感染原以及相关细胞信号转导过程中起到重要作用,同时还参与有丝分裂的应答过程；但是当体内活性氧簇过量时,由于氧自由基会对生物体造成伤害,因此会引发氧化应激。氧化应激反应(oxidative stress response)就是指当细胞内的活性氧簇水平高于细胞的抗氧化能力时产生的适应性反应。氧化应激与多种病理过程相关,包括心血管疾病、癌症、神经紊乱、糖尿病等,也是衰老的罪魁祸首。通常,抗氧化酶或其他抗氧化物缺陷时会发生氧化应激,即当机体产氧与抗氧化反应失衡时即发生氧化应激。
研究已经证实,高浓度的活性氧簇会对细胞结构以及核酸、脂质和蛋白质等造成损伤。其中,羟自由基(·OH)可以与DNA分子的所有组份发生反应,导致嘌呤,嘧啶以及脱氧核糖骨架的损伤；金属诱导产生的活性氧簇不仅攻击DNA,还会对氧化敏感的磷脂不饱和脂肪酸残基造成损伤；活性氧簇还能够对暴露于羟自由基或者羟/超氧化物自由基混合物中的氨基酸、多肽和蛋白质进行离子辐射,从而导致对蛋白质的氧化损伤。
由于机体经常暴露于各种来源的自由基、活性氧簇中,为了保持细胞内氧化还原的稳定状态,机体内产生了一系列的抗氧化机制,包括预防机制、修复机制、物理防卫和抗氧化防卫等。在有机体的主要抗氧化系统中,谷胱甘肽和硫氧还蛋白(thioredoxin,TRX)在细胞内的氧化还原动态平衡中起到了氧化还原缓冲的作用。因此,细胞内的稳态实际上就是由活性氧簇的产生速率与各种抗氧化物对其的清除速率决定的,维持活性氧簇的平衡,即氧化还原调控,成为机体内维持氧化还原稳态的一个非常重要的方面。
氧化还原调控过程就是保护机体免受各种氧化应激反应,并且通过控制氧化还原状态从而维持体内氧化还原的动态平衡。一系列研究表明在真核细胞中,有丝分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导通路在细胞氧化应激反应中起到关键作用。已知的MAPK家族包括细胞外调节蛋白激酶(extracellular-regulated kinases, ERK), c-Jun氨基末端激酶(c-jun-NH2-terminal kinases, JNK), p38MAPK和大丝裂原活化蛋白激酶1 (big MAPK-1, BMK-1).尼克酰胺腺嘌呤二核苷酸磷酸氧化酶1(NADPH oxidase 1, NOX1)功能产物、过氧化物和过氧化氢等氧化产物均可激活MAPK在MEK和ERK1/2水平上的信号级联。有研究表明,MAPK氧化应激过程中信号转导通路的激活是类型特异性和刺激特异性的,内源性过氧化氢激活ERK而不激活p38激酶；相反,外源性的过氧化氢激活p38激酶而非ERK。
PRAK (p38 regulated/activated kinase)即MK5,发现于1998年,为MAPK信号转导通路中一种受p38调控的丝氨酸/苏氨酸蛋白激酶。PRAK与其他受p38调控的MK族蛋白均存在核定位和核输出信号,但是特别的是,PRAK的两个定位信号有部分序列重叠,提示PRAK蛋白在细胞内定位方面有特殊的调控机制,也提示其在功能方面相比于其他MKs存在特殊之处。目前对PRAK功能方面的研究较少,根据已有实验表明,在受到外源性过氧化氢刺激时PRAK-/-型小鼠胚胎成纤维细胞(mouse embryo fibroblasts, MEF)凋亡率明显高于PRAK+/+型细胞,提示PRAK蛋白在细胞氧化应激反应过程中扮演着重要的角色。但是上述作用的具体机制尚不明确。因此,本实验的目的即在于发现细胞氧化应激过程中受到PRAK磷酸化调控的蛋白质,从而揭示PRAK蛋白参与细胞氧化应激调节过程的具体途径。
传统的研究思路通常是研究者根据已有的工作基础和文献报道,通过分析并预测得到在特定过程中有可能与目的蛋白发生相互作用的一个或几个重要分子,进而进行深入细致的功能研究。但是这种传统研究策略应用于本研究却缺乏可行性,并存在着局限性。首先,目前对PRAK已有的研究报道较少,难以为分析预测提供充分可靠而有效的理论支持；其次,传统的研究结果仅仅局限于一个或几个蛋白,说明的科学问题也仅仅停留在“点”的阶段,很难从整体上水平上解释复杂的生命现象。不仅如此,机体内的蛋白质是处于是高度动态变化的,即使同一细胞在不同生理或病理环境中,其蛋白表达的时空差异性也是不同的,而这种差异蛋白往往恰是某些疾病发生的标志。基于上述的考虑,我们在研究过程中采用了蛋白质组学的研究策略。
蛋白质组学是独立于基因组学发展起来的一门新兴的前沿学科,它在特定的时间和空间研究一个完整的生物体或细胞所表达的全部蛋白质的特征,包括蛋白质的表达水平、翻译后修饰(post-translational modifications, PTMs)、蛋白质与蛋白质相互作用等,从而在蛋白质水平上获得对于生物体生理、病理等过程的全面认识,具有大规模、高通量、高灵敏度的特点。利用比较蛋白质组研究技术,我们可以批量观察细胞在正常与应激状态下蛋白质表达谱上的差异,从整体水平上规模化地筛选和发掘潜在的功能蛋白点,从而大大加快我们对蛋白质功能及其调节过程的认识。
由于整个细胞或组织的蛋白组成极其复杂,蛋白质组学的发展对于研究技术手段有着较高的要求,推动了多项前沿生物研究技术的革新与发展,其中就包括了荧光差异双向电泳技术(fluorescence difference gel electrophoresis, DIGE)。DIGE技术是经典双向电泳技术(two-dimensional gel electrophoresis,2-DE)的成熟与完善,通过三色荧光染料分别对内标和生物样本进行标记,能够在一张2-DE胶上对两个样本的多种蛋白质进行分离,并分别单独成像,应用DeCyder 2-DE软件分析图像,得到蛋白表达量的丰度变化,根据数据分析结果准确地发现差异蛋白。使用内标、正反标的实验设计有效消除胶内和胶间差异,结果准确、可靠、重复性好。全自动软件分析对蛋白质表达量进行校准,降低由实验因素、数据分析和生物种群间固有个体差异等引起的系统误差至最小,确保定性的准确度。
蛋白质磷酸化(protein phosphorylation)是生物界最普遍,也是最重要的一种蛋白质翻译后修饰方式,很多重要蛋白质的活性是通过翻译后修饰过程来进行调节的。可以预测,PRAK作为MAPK信号转导通路上的重要丝氨酸/苏氨酸蛋白激酶,它对下游蛋白的调控作用很可能也是通过使后者发生磷酸化来实现的。因此,在本课题研究中,我们采用定量技术分别对亚砷酸钠刺激前后PRAK+/+与PRAK-/-型小鼠胚胎成纤维细胞磷酸化蛋白质组学进行差异定量分析,以期寻找一批在氧化应激过程中受PRAK磷酸化调控的下游功能蛋白。该项工作的开展不仅能够帮助我们全面而深入的认识PRAK在氧化应激过程中所承担的细胞调节功能,并且对于阐明内源性抗氧化通路具有重要意义。
基于上述思考,本实验的研究内容为：
(1)分别用亚砷酸钠刺激(NaAsO2 45min)PRAK+/+与PRAK-/-型小鼠胚胎成纤维细胞；
(2)利用金属磷酸盐亲和层析树脂富集磷酸化蛋白；
(3)采用DIGE技术对磷酸化蛋白进行分离,并建立相应的差异蛋白图谱；
(4)通过DeCyder差异分析软件分析后找到差异蛋白点
(4)对这些差异蛋白点利用MALDI-TOF/TOF质谱仪进行蛋白质鉴定
(5)对鉴定出的蛋白质从功能,定位,相互作用等方面进行生物信息学分析。
综上所述,我们的研究得出以下几点结论：
1.应用金属磷酸盐亲和层析树脂成功富集了PRAK+/+和PRAK-/-小鼠胚胎成纤维细胞的胞质磷酸化蛋白。利用DIGE技术成功分离并建立了相应的差异蛋白图谱。通过相应的软件分析,得到了32个具有统计学意义的差异蛋白点；
2.对32个差异蛋白点进行质谱鉴定,去除角蛋白污染和重复蛋白后,共鉴定出16种蛋白,其中发现有13种蛋白为已经确证的磷酸化蛋白质；
3.对16种差异蛋白进行亚细胞定位分析发现,定位于胞浆的有8个；1个蛋白完全定位于细胞膜；1个蛋白完全定位于线粒体；3个蛋白分泌到胞外；3个蛋白可能存在胞浆与胞核间的穿梭；
4.通过生物信息学分析发现这些差异蛋白包括膜蛋白,离子通道蛋白,细胞骨架蛋白,氧化还原酶,细胞因子；说明氧化应激应答过程中PRAK通过磷酸化调控细胞功能的多个方面。
5. String软件预测鉴定的差异蛋白,其中彼此有相互作用的较少,可能是由于质谱鉴定丢失了部分差异点造成,也可能是由于目前对PRAK已有的研究较少,无法为相互作用的预测提供充足的依据。
6.分析这16种蛋白在两组细胞中的刺激前后磷酸化水平变化情况,发现氧化应激过程中,有些蛋白直接受到PRAK调控磷酸化水平上调,在PRAK+/+细胞刺激后磷酸化水平明显上调而在PRAK-/-细胞刺激后变化不明显；与上述情况相反,少量蛋白在PRAK-/-细胞刺激后磷酸化水平上调明显,推测可能受到PRAK代偿通路的调控；个别蛋白在刺激后磷酸化水平反而下降,可能是PRAK通过对磷酸酶的调控而间接影响其磷酸化水平的。
Oxygen free. radicals or, more generally, reactive oxygen species (ROS), including·O2,·OH, H2O2, NO-, are products of normal cellular metabolism. ROS is well recognised for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. When the ROS is at low/moderate concentrations, beneficial effects of ROS occur, as for example in defence against infectious agents and in the function of a number of cellular signaling systems. One further beneficial example of ROS at low/moderate concentrations is the induction of a mitogenic response. But if the free radicals is excessive, the potential biological damage is termed oxidative stress. Oxidative stress has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological disorders, diabetes, ischemia/reperfusion, other diseases and ageing.
The excess ROS can damage cellular lipids, proteins, or DNA inhibiting their normal function. Because of this, oxidative stress has been implicated in a number of human diseases as well as in the ageing process. The hydroxyl radical is known to react with all components of the DNA molecule, leading to both the purine and pyrimidine bases damaged and also the deoxyribose backbone. It is known that metal-induced generation of ROS results in an attack not only on DNA, but also on other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation. Mechanisms involved in the oxidation of proteins by ROS were elucidated by studies in which amino acids, simple peptides and proteins were exposed to ionizing radiations under conditions where hydroxyl radicals or a mixture of hydroxyl/superoxide radicals are formed. The side chains of all amino acid residues of proteins, in particular cysteine and methionine residues of proteins are susceptible to oxidation by the action of ROS.
Because the body is constantly exposed to a variety of sources of free radicals, reactive oxygen species, in order to maintain the stability of the intracellular redox state, the body produces a series of anti-oxidation mechanism, involve:preventative mechanisms, repair mechanisms, physical defences, and antioxidant defences. Their "steady state" concentrations are determined by the balance between their rates of production and their rates of removal by various antioxidants.
The delicate balance between beneficial and harmful effects of free radicals is a very important aspect of living organisms and is achieved by mechanisms called "redox regulation. The process of "redox regulation" protects living organisms from various oxidative stresses and maintains "redox homeostasis" by controlling the redox status in vivo.
A number of studies reported that the serine/threonine kinases of the MAPK family can be regulated by oxidants. There are four known MAPK families:extracellular-regulated (ERKs), c-jun-NH2-terminal kinase (JNKs), p38 MAPK and the big MAPK-1 (BMK-1), of which serine/thereonine kinases are important in the process of carcinogenesis including cell proliferation, differentiation and apoptosis. Products of NOX1 activity, superoxide, hydrogen peroxide can activate the MAPK cascade at the level of MEK and ERK1/2. The experimental studies on the up-regulation of MAPKs by H2O2 treatment have shown that the activation of each signaling pathway is type-and stimulus-specific. For example, it has been reported that endogenous H2O2 production by the respiratory burst induces ERK but not p38 kinase activity. Conversely, exogenous H2O2 activates p38 kinase, but not ERK in rat alvedor macrophages. The ERK pathway has most commonly been associated with the regulation of cell proliferation. The balance between ERK and JNK activation is a key factor for cell survival since both a decrease in ERK and an increase in JNK are required for the induction of apoptosis.
PRAK (p38-regulated and-activated kinase), also known as MK5, first discovered in 1998, is an important serine/threonine protein kinase regulated by p38 in the downstream of the MAPK signal transduction pathway. PRAK and other MKs all have nuclear localization and nuclear export signal, but what in particular is that the two localization sequences of PRAK overlap, suggesting its localization in the cell has a special regulatory mechanism and also it has some special function compared with other MKs. Previous experiment showed that PRAK plays an important role in the cell during oxidative stress. When stimulated by exogenous hydrogen peroxide the death rate of PRAK-/- cells was significantly higher than PRAK+/+ cells. But the specific mechanism is still not clear. Therefore, this study was designed to reveal the protein phosphorylated by PRAK in the process of cellular oxidative stress.
According to traditional research methods which based on the existing work of PRAK, some molecules interacting with PRAK in the process of oxidative stress response could be derived. And with a detailed study we can find one or several important molecules. But this traditional research strategy is difficult to achieve. First, there is less research to provide reliable and effective for the analysis of PRAK currently; Second, this study, focusing on one or several proteins,is difficult to explain the whole problem. Only when this "point" of the results are accumulating enough, the system's problems will become gradually clear up, but this process generally takes a long time.
The proteome is highly dynamic, even if the same cells in different physiological or pathological conditions, the expression of the highly dynamic proteome is different, and this difference protein often just a sign of some disease. Using comparative proteomics research techniques, we can observe differences of the protein expression profile in normal and disease cells (tissue), screen and explore potential functions proteins and markers for early diagnosis, intervention and treatment of disease.
As a large-scale, high throughput, high sensitivity method, proteomics can effectively analyze the overall intracellular proteins. However, the protein composition of cells or tissues is so extremely complex that the development of proteomics is restricted and driven by the technology. The study of Proteomics depends largely on the level of their skill level.
Fluorescence differential gel electrophoresis (DIGE) is a method which labels protein samples with different fluorescent dyes before 2-D electrophoresis, and then to three different protein samples are separated up at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the biological differences really, while avoid influence of systematic errors. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye multiple labeling method and DeCyder difference 2-D analysis software. DeCyder software takes the advantage of the spots co-detecting algorithm, which can automatically detected fluorescence images, eliminate background, quantify, normalize and match spots in gel, thus, systematic errors caused by different operators can be eliminated.
Protein phosphorylation is the most common as well as the most important type of protein post-translational modification. The activity of many proteins is regulated by the post-translational modification, particularly PRAK as the important serine/threonine protein kinase in MAPK signal transduction pathway, its regulation of the downstream proteins be achieved by phosphorylation. So looking for the proteins regulated by PRAK at the process of oxidative stress is helpful for not only Comprehensive and in-depth understanding of PRAK, but also Searching for effective endogenous antioxidant pathway.
Based on such considerations, we design a differential proteomics experiments by stimulated normal and PRAK-/- MEF cells 45min with NaAsO2. Then cytoplasm phosphoproteins were extracted by the phosphate metal affinity chromatography resin (PMAC). Finally, the differential expression of protein profile of four groups has been established by using DIGE technology. Through analysis by use of DeCyder difference 2-D analysis software, 32 differential protein spots were found. The numbers of up-regulated proteins is 18 in the normal MEF cells after NaAsO2 stimulated and 11 in the PRAK-/- cells; while the numbers of down-regulated proteins is 2 in the normal MEF cells after stimulated and 1 in the PRAK-/ cells. At last, a total of 16 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF). 13 of them are confirmed to be phosphorylated.
We predicted the subcellular localization of the differential proteins and made a result that, most proteins locate in cytoplasm and nucleur. After subcellular localization analysis, the protein functional analysis has been done. Through analyzing protein domain and motif database, the identified proteins roughly included Membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines. However, we also predicted protein interactions using the "String" protein interaction databases. We found that Interacting proteins were foundless.
Based on the researches above, we have some conclusion:
1. Cytoplasm phosphoproteins were enriched through the phosphate metal affinity chromatography resin (PMAC). Subsequently, the phosphoproteins of control and stimulated (NaAsO2 45min) normal and PRAK-/- MEF cells were separated by DIGE.32 differential protein spots with statistical significance were detected.
2. The significant differential protein spots were analyzed by mass spectrometry and 16 proteins were identified after deleting keratin and duplication proteins.13 of them have had confirmed to be phosphorylated.
3. We predicted the subcellular localization of the differential proteins and made a result that most proteins locate in cytoplasm and nucleur.
4. Through analyzing protein domain and motif database, the identified proteins roughly included membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines.
5. We also predicted protein interactions using the "String" protein interaction databases. The result that interacting proteins were foundless, shows the process of oxidative stress regulated by PRAK involved many aspects of cell function, rather than a complex of proteins share the same function.
6. Analysis the phosphorylation level of these 16 proteins in different cells and found that in the process of oxidative stress, some proteins directly regulated by PRAK, and a small amount of proteins may be regulated through a compensatory pathway when there is no PRAK expressed and some proteins may be regulated through specific protein which phosphorylated by PRAK.
研究已经证实,高浓度的活性氧簇会对细胞结构以及核酸、脂质和蛋白质等造成损伤。其中,羟自由基(·OH)可以与DNA分子的所有组份发生反应,导致嘌呤,嘧啶以及脱氧核糖骨架的损伤；金属诱导产生的活性氧簇不仅攻击DNA,还会对氧化敏感的磷脂不饱和脂肪酸残基造成损伤；活性氧簇还能够对暴露于羟自由基或者羟/超氧化物自由基混合物中的氨基酸、多肽和蛋白质进行离子辐射,从而导致对蛋白质的氧化损伤。
由于机体经常暴露于各种来源的自由基、活性氧簇中,为了保持细胞内氧化还原的稳定状态,机体内产生了一系列的抗氧化机制,包括预防机制、修复机制、物理防卫和抗氧化防卫等。在有机体的主要抗氧化系统中,谷胱甘肽和硫氧还蛋白(thioredoxin,TRX)在细胞内的氧化还原动态平衡中起到了氧化还原缓冲的作用。因此,细胞内的稳态实际上就是由活性氧簇的产生速率与各种抗氧化物对其的清除速率决定的,维持活性氧簇的平衡,即氧化还原调控,成为机体内维持氧化还原稳态的一个非常重要的方面。
氧化还原调控过程就是保护机体免受各种氧化应激反应,并且通过控制氧化还原状态从而维持体内氧化还原的动态平衡。一系列研究表明在真核细胞中,有丝分裂原活化蛋白激酶(mitogen-activated protein kinases, MAPK)信号转导通路在细胞氧化应激反应中起到关键作用。已知的MAPK家族包括细胞外调节蛋白激酶(extracellular-regulated kinases, ERK), c-Jun氨基末端激酶(c-jun-NH2-terminal kinases, JNK), p38MAPK和大丝裂原活化蛋白激酶1 (big MAPK-1, BMK-1).尼克酰胺腺嘌呤二核苷酸磷酸氧化酶1(NADPH oxidase 1, NOX1)功能产物、过氧化物和过氧化氢等氧化产物均可激活MAPK在MEK和ERK1/2水平上的信号级联。有研究表明,MAPK氧化应激过程中信号转导通路的激活是类型特异性和刺激特异性的,内源性过氧化氢激活ERK而不激活p38激酶；相反,外源性的过氧化氢激活p38激酶而非ERK。
PRAK (p38 regulated/activated kinase)即MK5,发现于1998年,为MAPK信号转导通路中一种受p38调控的丝氨酸/苏氨酸蛋白激酶。PRAK与其他受p38调控的MK族蛋白均存在核定位和核输出信号,但是特别的是,PRAK的两个定位信号有部分序列重叠,提示PRAK蛋白在细胞内定位方面有特殊的调控机制,也提示其在功能方面相比于其他MKs存在特殊之处。目前对PRAK功能方面的研究较少,根据已有实验表明,在受到外源性过氧化氢刺激时PRAK-/-型小鼠胚胎成纤维细胞(mouse embryo fibroblasts, MEF)凋亡率明显高于PRAK+/+型细胞,提示PRAK蛋白在细胞氧化应激反应过程中扮演着重要的角色。但是上述作用的具体机制尚不明确。因此,本实验的目的即在于发现细胞氧化应激过程中受到PRAK磷酸化调控的蛋白质,从而揭示PRAK蛋白参与细胞氧化应激调节过程的具体途径。
传统的研究思路通常是研究者根据已有的工作基础和文献报道,通过分析并预测得到在特定过程中有可能与目的蛋白发生相互作用的一个或几个重要分子,进而进行深入细致的功能研究。但是这种传统研究策略应用于本研究却缺乏可行性,并存在着局限性。首先,目前对PRAK已有的研究报道较少,难以为分析预测提供充分可靠而有效的理论支持；其次,传统的研究结果仅仅局限于一个或几个蛋白,说明的科学问题也仅仅停留在“点”的阶段,很难从整体上水平上解释复杂的生命现象。不仅如此,机体内的蛋白质是处于是高度动态变化的,即使同一细胞在不同生理或病理环境中,其蛋白表达的时空差异性也是不同的,而这种差异蛋白往往恰是某些疾病发生的标志。基于上述的考虑,我们在研究过程中采用了蛋白质组学的研究策略。
蛋白质组学是独立于基因组学发展起来的一门新兴的前沿学科,它在特定的时间和空间研究一个完整的生物体或细胞所表达的全部蛋白质的特征,包括蛋白质的表达水平、翻译后修饰(post-translational modifications, PTMs)、蛋白质与蛋白质相互作用等,从而在蛋白质水平上获得对于生物体生理、病理等过程的全面认识,具有大规模、高通量、高灵敏度的特点。利用比较蛋白质组研究技术,我们可以批量观察细胞在正常与应激状态下蛋白质表达谱上的差异,从整体水平上规模化地筛选和发掘潜在的功能蛋白点,从而大大加快我们对蛋白质功能及其调节过程的认识。
由于整个细胞或组织的蛋白组成极其复杂,蛋白质组学的发展对于研究技术手段有着较高的要求,推动了多项前沿生物研究技术的革新与发展,其中就包括了荧光差异双向电泳技术(fluorescence difference gel electrophoresis, DIGE)。DIGE技术是经典双向电泳技术(two-dimensional gel electrophoresis,2-DE)的成熟与完善,通过三色荧光染料分别对内标和生物样本进行标记,能够在一张2-DE胶上对两个样本的多种蛋白质进行分离,并分别单独成像,应用DeCyder 2-DE软件分析图像,得到蛋白表达量的丰度变化,根据数据分析结果准确地发现差异蛋白。使用内标、正反标的实验设计有效消除胶内和胶间差异,结果准确、可靠、重复性好。全自动软件分析对蛋白质表达量进行校准,降低由实验因素、数据分析和生物种群间固有个体差异等引起的系统误差至最小,确保定性的准确度。
蛋白质磷酸化(protein phosphorylation)是生物界最普遍,也是最重要的一种蛋白质翻译后修饰方式,很多重要蛋白质的活性是通过翻译后修饰过程来进行调节的。可以预测,PRAK作为MAPK信号转导通路上的重要丝氨酸/苏氨酸蛋白激酶,它对下游蛋白的调控作用很可能也是通过使后者发生磷酸化来实现的。因此,在本课题研究中,我们采用定量技术分别对亚砷酸钠刺激前后PRAK+/+与PRAK-/-型小鼠胚胎成纤维细胞磷酸化蛋白质组学进行差异定量分析,以期寻找一批在氧化应激过程中受PRAK磷酸化调控的下游功能蛋白。该项工作的开展不仅能够帮助我们全面而深入的认识PRAK在氧化应激过程中所承担的细胞调节功能,并且对于阐明内源性抗氧化通路具有重要意义。
基于上述思考,本实验的研究内容为：
(1)分别用亚砷酸钠刺激(NaAsO2 45min)PRAK+/+与PRAK-/-型小鼠胚胎成纤维细胞；
(2)利用金属磷酸盐亲和层析树脂富集磷酸化蛋白；
(3)采用DIGE技术对磷酸化蛋白进行分离,并建立相应的差异蛋白图谱；
(4)通过DeCyder差异分析软件分析后找到差异蛋白点
(4)对这些差异蛋白点利用MALDI-TOF/TOF质谱仪进行蛋白质鉴定
(5)对鉴定出的蛋白质从功能,定位,相互作用等方面进行生物信息学分析。
综上所述,我们的研究得出以下几点结论：
1.应用金属磷酸盐亲和层析树脂成功富集了PRAK+/+和PRAK-/-小鼠胚胎成纤维细胞的胞质磷酸化蛋白。利用DIGE技术成功分离并建立了相应的差异蛋白图谱。通过相应的软件分析,得到了32个具有统计学意义的差异蛋白点；
2.对32个差异蛋白点进行质谱鉴定,去除角蛋白污染和重复蛋白后,共鉴定出16种蛋白,其中发现有13种蛋白为已经确证的磷酸化蛋白质；
3.对16种差异蛋白进行亚细胞定位分析发现,定位于胞浆的有8个；1个蛋白完全定位于细胞膜；1个蛋白完全定位于线粒体；3个蛋白分泌到胞外；3个蛋白可能存在胞浆与胞核间的穿梭；
4.通过生物信息学分析发现这些差异蛋白包括膜蛋白,离子通道蛋白,细胞骨架蛋白,氧化还原酶,细胞因子；说明氧化应激应答过程中PRAK通过磷酸化调控细胞功能的多个方面。
5. String软件预测鉴定的差异蛋白,其中彼此有相互作用的较少,可能是由于质谱鉴定丢失了部分差异点造成,也可能是由于目前对PRAK已有的研究较少,无法为相互作用的预测提供充足的依据。
6.分析这16种蛋白在两组细胞中的刺激前后磷酸化水平变化情况,发现氧化应激过程中,有些蛋白直接受到PRAK调控磷酸化水平上调,在PRAK+/+细胞刺激后磷酸化水平明显上调而在PRAK-/-细胞刺激后变化不明显；与上述情况相反,少量蛋白在PRAK-/-细胞刺激后磷酸化水平上调明显,推测可能受到PRAK代偿通路的调控；个别蛋白在刺激后磷酸化水平反而下降,可能是PRAK通过对磷酸酶的调控而间接影响其磷酸化水平的。
Oxygen free. radicals or, more generally, reactive oxygen species (ROS), including·O2,·OH, H2O2, NO-, are products of normal cellular metabolism. ROS is well recognised for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. When the ROS is at low/moderate concentrations, beneficial effects of ROS occur, as for example in defence against infectious agents and in the function of a number of cellular signaling systems. One further beneficial example of ROS at low/moderate concentrations is the induction of a mitogenic response. But if the free radicals is excessive, the potential biological damage is termed oxidative stress. Oxidative stress has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological disorders, diabetes, ischemia/reperfusion, other diseases and ageing.
The excess ROS can damage cellular lipids, proteins, or DNA inhibiting their normal function. Because of this, oxidative stress has been implicated in a number of human diseases as well as in the ageing process. The hydroxyl radical is known to react with all components of the DNA molecule, leading to both the purine and pyrimidine bases damaged and also the deoxyribose backbone. It is known that metal-induced generation of ROS results in an attack not only on DNA, but also on other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation. Mechanisms involved in the oxidation of proteins by ROS were elucidated by studies in which amino acids, simple peptides and proteins were exposed to ionizing radiations under conditions where hydroxyl radicals or a mixture of hydroxyl/superoxide radicals are formed. The side chains of all amino acid residues of proteins, in particular cysteine and methionine residues of proteins are susceptible to oxidation by the action of ROS.
Because the body is constantly exposed to a variety of sources of free radicals, reactive oxygen species, in order to maintain the stability of the intracellular redox state, the body produces a series of anti-oxidation mechanism, involve:preventative mechanisms, repair mechanisms, physical defences, and antioxidant defences. Their "steady state" concentrations are determined by the balance between their rates of production and their rates of removal by various antioxidants.
The delicate balance between beneficial and harmful effects of free radicals is a very important aspect of living organisms and is achieved by mechanisms called "redox regulation. The process of "redox regulation" protects living organisms from various oxidative stresses and maintains "redox homeostasis" by controlling the redox status in vivo.
A number of studies reported that the serine/threonine kinases of the MAPK family can be regulated by oxidants. There are four known MAPK families:extracellular-regulated (ERKs), c-jun-NH2-terminal kinase (JNKs), p38 MAPK and the big MAPK-1 (BMK-1), of which serine/thereonine kinases are important in the process of carcinogenesis including cell proliferation, differentiation and apoptosis. Products of NOX1 activity, superoxide, hydrogen peroxide can activate the MAPK cascade at the level of MEK and ERK1/2. The experimental studies on the up-regulation of MAPKs by H2O2 treatment have shown that the activation of each signaling pathway is type-and stimulus-specific. For example, it has been reported that endogenous H2O2 production by the respiratory burst induces ERK but not p38 kinase activity. Conversely, exogenous H2O2 activates p38 kinase, but not ERK in rat alvedor macrophages. The ERK pathway has most commonly been associated with the regulation of cell proliferation. The balance between ERK and JNK activation is a key factor for cell survival since both a decrease in ERK and an increase in JNK are required for the induction of apoptosis.
PRAK (p38-regulated and-activated kinase), also known as MK5, first discovered in 1998, is an important serine/threonine protein kinase regulated by p38 in the downstream of the MAPK signal transduction pathway. PRAK and other MKs all have nuclear localization and nuclear export signal, but what in particular is that the two localization sequences of PRAK overlap, suggesting its localization in the cell has a special regulatory mechanism and also it has some special function compared with other MKs. Previous experiment showed that PRAK plays an important role in the cell during oxidative stress. When stimulated by exogenous hydrogen peroxide the death rate of PRAK-/- cells was significantly higher than PRAK+/+ cells. But the specific mechanism is still not clear. Therefore, this study was designed to reveal the protein phosphorylated by PRAK in the process of cellular oxidative stress.
According to traditional research methods which based on the existing work of PRAK, some molecules interacting with PRAK in the process of oxidative stress response could be derived. And with a detailed study we can find one or several important molecules. But this traditional research strategy is difficult to achieve. First, there is less research to provide reliable and effective for the analysis of PRAK currently; Second, this study, focusing on one or several proteins,is difficult to explain the whole problem. Only when this "point" of the results are accumulating enough, the system's problems will become gradually clear up, but this process generally takes a long time.
The proteome is highly dynamic, even if the same cells in different physiological or pathological conditions, the expression of the highly dynamic proteome is different, and this difference protein often just a sign of some disease. Using comparative proteomics research techniques, we can observe differences of the protein expression profile in normal and disease cells (tissue), screen and explore potential functions proteins and markers for early diagnosis, intervention and treatment of disease.
As a large-scale, high throughput, high sensitivity method, proteomics can effectively analyze the overall intracellular proteins. However, the protein composition of cells or tissues is so extremely complex that the development of proteomics is restricted and driven by the technology. The study of Proteomics depends largely on the level of their skill level.
Fluorescence differential gel electrophoresis (DIGE) is a method which labels protein samples with different fluorescent dyes before 2-D electrophoresis, and then to three different protein samples are separated up at the same time in one two-dimensional gel. The application of the internal standard could further increase the credibility of the experiment, and ensure the results could reflect the biological differences really, while avoid influence of systematic errors. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye multiple labeling method and DeCyder difference 2-D analysis software. DeCyder software takes the advantage of the spots co-detecting algorithm, which can automatically detected fluorescence images, eliminate background, quantify, normalize and match spots in gel, thus, systematic errors caused by different operators can be eliminated.
Protein phosphorylation is the most common as well as the most important type of protein post-translational modification. The activity of many proteins is regulated by the post-translational modification, particularly PRAK as the important serine/threonine protein kinase in MAPK signal transduction pathway, its regulation of the downstream proteins be achieved by phosphorylation. So looking for the proteins regulated by PRAK at the process of oxidative stress is helpful for not only Comprehensive and in-depth understanding of PRAK, but also Searching for effective endogenous antioxidant pathway.
Based on such considerations, we design a differential proteomics experiments by stimulated normal and PRAK-/- MEF cells 45min with NaAsO2. Then cytoplasm phosphoproteins were extracted by the phosphate metal affinity chromatography resin (PMAC). Finally, the differential expression of protein profile of four groups has been established by using DIGE technology. Through analysis by use of DeCyder difference 2-D analysis software, 32 differential protein spots were found. The numbers of up-regulated proteins is 18 in the normal MEF cells after NaAsO2 stimulated and 11 in the PRAK-/- cells; while the numbers of down-regulated proteins is 2 in the normal MEF cells after stimulated and 1 in the PRAK-/ cells. At last, a total of 16 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/TOF). 13 of them are confirmed to be phosphorylated.
We predicted the subcellular localization of the differential proteins and made a result that, most proteins locate in cytoplasm and nucleur. After subcellular localization analysis, the protein functional analysis has been done. Through analyzing protein domain and motif database, the identified proteins roughly included Membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines. However, we also predicted protein interactions using the "String" protein interaction databases. We found that Interacting proteins were foundless.
Based on the researches above, we have some conclusion:
1. Cytoplasm phosphoproteins were enriched through the phosphate metal affinity chromatography resin (PMAC). Subsequently, the phosphoproteins of control and stimulated (NaAsO2 45min) normal and PRAK-/- MEF cells were separated by DIGE.32 differential protein spots with statistical significance were detected.
2. The significant differential protein spots were analyzed by mass spectrometry and 16 proteins were identified after deleting keratin and duplication proteins.13 of them have had confirmed to be phosphorylated.
3. We predicted the subcellular localization of the differential proteins and made a result that most proteins locate in cytoplasm and nucleur.
4. Through analyzing protein domain and motif database, the identified proteins roughly included membrane protein, oxidoreductase, Ion channel protein, Cytoskeletal proteins, Cytokines.
5. We also predicted protein interactions using the "String" protein interaction databases. The result that interacting proteins were foundless, shows the process of oxidative stress regulated by PRAK involved many aspects of cell function, rather than a complex of proteins share the same function.
6. Analysis the phosphorylation level of these 16 proteins in different cells and found that in the process of oxidative stress, some proteins directly regulated by PRAK, and a small amount of proteins may be regulated through a compensatory pathway when there is no PRAK expressed and some proteins may be regulated through specific protein which phosphorylated by PRAK.
引文
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