固醇携带蛋白2（SCP2）和植烷酸氧化酶（Phyh）相互作用的鉴定

发布时间：2018-02-10 01:13

本文关键词： 蛋白质相互作用植烷酸氧化酶固醇携带蛋白　出处：《南方医科大学》2010年硕士论文　论文类型：学位论文

【摘要】：生物体内生命携带的遗传信息由不同的基因经转录、翻译传递到相应的蛋白质上并使其具有各自的生化特性及生物学活性；但每个蛋白质并不是独立地在细胞中完成被赋予的功能,它们在细胞中通常与其他蛋白质相互作用形成大的复合体,在特定的时间和空间内完成特定的功能,而且有些蛋白质的功能只有在复合体形成后才能发挥出来。蛋白质间的相互作用可改变细胞内蛋白质的动力学特征,如底物结合特性、催化活性；也可产生新的结合位点,改变蛋白质对底物的特异性；还可使其它蛋白质失活,调控其它基因表达。只有蛋白质间相互作用顺利进行,细胞的正常生命活动过程才有保障。因此,研究蛋白质相互作用,对于深入了解蛋白质的功能,阐明生命活动的规律有着重要的意义。目前用于研究蛋白质间相互作用的方法包括酵母双杂交(yeast-two hybrid)、体外结合实验(in vitro binding)、免疫共沉淀(coimmunoprecipitation)、串联亲和纯化(tandem affinity purification, TAP)、质谱鉴定(mass spectrometry)、蛋白芯片(protein chip)以及基于生物信息学(Bioinformatics)的分析方法等。其中,生物信息学分析无需具体的实验操作,往往是在整个基因组规模上进行研究,可获得较大的信息量。另外根据蛋白晶体结构的互补性,也可以推测蛋白间可能的相互作用。因此,生物信息学分析为研究蛋白质相互作用的方法提供了一个新的方向,为实验提供了重要的线索。本实验室前期利用差异凝胶电泳(differential in-gel electrophoresis, DIGE)和质谱分析技术鉴定出内毒素休克小鼠线粒体内22种含量发生改变的蛋白质,并运用生物信息学技术进行了蛋白质相互作用预测,结果找到了一组具有相互作用的蛋白-蛋白复合物：固醇携带蛋白2(sterol carrier protein 2, SCP2)／植烷酸氧化酶(phytanoyl-CoA hydroxylase, Phyh)。实验发现在脂多糖(lipopolysaccharide, LPS)刺激小鼠1 hr后线粒体内SCP2和Phyh的含量同时减少,并且减少倍数一致,因此,可以推测它们可能是两者或与其他蛋白一起以功能复合物的形式通过某种通道穿梭出线粒体,从而参与机体对内毒素的应激反应。另外,通过对这两个蛋白进行分析我们发现,两者存在一些共性,例如两者在线粒体中均有定位,且都有参与脂肪酸代谢。在上述基础上,我们确立了此项研究,即运用相关实验方法来验证SCP2/Phyh这对小鼠线粒体蛋白质之间的相互作用。如果实验证明这两个蛋白确实存在相互作用,就可以提示我们两者是以复合物的形式参与脂肪酸代谢,这可以为我们研究与这两个蛋白相关的一些代谢性疾病提供新的方向。 Phyh蛋白主要定位于过氧化物酶体和线粒体中。它负责催化过氧化物酶体α-氧化途径的首个反应,即将植烷酰辅酶A转化为2-羟基植烷酸辅酶A。先前的研究发现,当Phyh基因发生突变时,可引起植烷酸氧化障碍,进而导致机体肾脏和神经系统的一系列症状的产生。目前对该蛋白的研究多集中在Phyh基因的突变与雷弗素姆病的关系上。迄今为止,国外已经陆续报道了几种Phyh基因的突变,例如Arg275Trp,即第275位的精氨酸被色氨酸取代。突变的Phyh失去氧化植烷酸的活性,进而导致植烷酸代谢障碍,大量植烷酸蓄积于各种组织和体液中,最终引起雷弗素姆病(Refsum's disease)、脑肝肾综合征(ceribro-hepato-renal syndrome)、肢近端型斑点状软骨发育不良(rhizomelic chondrodysplasi)和新生儿肾上腺白质营养不良症(neonatal adrenoleukodystrophy, NALD)等过氧化物酶体相关性代谢性疾病。此外,还有研究发现Phyh蛋白可以和一些蛋白发生相互作用,进而导致雷弗素姆病的发生,例如脑组织中的PAHX-AP#1蛋白可与Phyh蛋白发生相互作用,并与雷弗素姆病中枢神经系统方面的症状有一定的联系。 SCP2主要存在于线粒体、过氧化物酶体、内质网和胞质中,肝脏含量最高。它一方面作为胆固醇代谢的调节因子,参与胆固醇的生物合成和胆固醇向胆汁酸、胆固醇酯及类固醇激素的转化；另一方面作为胆固醇转运器,参与细胞内、质膜间胆固醇的运输,并能将肝脏新合成的胆固醇直接从内质网快速转运至胆汁。大量研究表明SCP2表达水平的升高是胆固醇结石发生的重要原因。为了验证Phyh和SCP2两者是否具有相互作用,我们从体内和体外分别验证了两者的相互作用。首先,我们构建了Phyh的原核和真核表达质粒,分别为pGEX-4T-Phyh和pcDNA3-HA-Phyh;SCP2的原核和真核表达质粒,分别为pET14b-SCP2和pcDNA3-Flag-SCP2。紧接着,为了研究两者在体外环境下是否存在相互作用,我们进行了体外结合实验,结果发现两者在体外可以特异性地结合。然后,我们又从体内进行了验证。先利用免疫荧光技术观察两个蛋白在线粒体的表达和共定位情况。结果发现两者均能定位于线粒体中,且存在共定位。而后运用免疫共沉淀技术观察两者的体内结合情况,结果在正常情况下并未能检测到两者在NIH/3T3细胞中的结合。于是设想两者仅在某些应激刺激下才发生相互作用,因此,为了获得确切的实验结果,我们进一步改进了实验方法和条件,增设了分别用NaAsO2和LPS刺激NIH/3T3细胞1h这两个实验组,结果仍未检测到两者的结合。对此结果进行分析,我们认为可能是由于细胞内环境比较复杂,Phyh和SCP2两者只有在某种特殊情况下,在胞内其他物质的作用下才会发生相互作用；或者是两者的相互作用很弱且短暂,通过当前的实验难以验证。究竟两者是否存在这种相互作用,还有待更深入的研究。综上所述,本研究为了验证Phyh和SCP2之间是否存在相互作用,首先构建了两者的原核和真核表达质粒,之后通过体外结合实验验证了两者在体外的相互作用,然后通过细胞免疫荧光技术观察两个蛋白在细胞内的共定位情况,最后通过免疫共沉淀实验来验证两者在体内的相互作用。通过以上研究,我们得到以下结论： 1.体外结合实验表明Phyh和SCP2在体外环境中能特异性地结合。 2.细胞免疫荧光发现Phyh和SCP2在线粒体中有共定位。 3.免疫共沉淀实验表明Phyh和SCP2在体内不能特异性地结合,两者是否在体内具有相互作用还需进行进一步的研究。本课题的研究结果丰富了蛋白质相互作用的信息,并为进行新的蛋白质相互作用的研究提供了新的思路和方法。
[Abstract]:The genetic information in life carried by different genes by transcription, translation is transferred to the corresponding protein which has biochemical characteristics and biological activity of each other; but each protein is not independent in the cells perform their function in cells usually interact with other proteins to form complexes. To perform a specific function in the specific time and space, and some only in the function of a protein complex formation can play. Protein protein interactions can change the dynamic characteristics of the protein in the cell, such as substrate binding properties, catalytic activity; also can produce new binding sites, the change of specific protein on the substrate; also can cause other protein inactivation, regulating expression of other genes. Only protein-protein interaction smoothly, the normal life activities of the cell process Therefore, the study of protein interaction is of great significance for understanding the function of protein and clarifying the law of life activities.
At present, the methods used to study the interaction between proteins including yeast two hybrid (yeast-two hybrid), in vitro binding assay (in vitro binding), CO immunoprecipitation (coimmunoprecipitation), tandem affinity purification (tandem affinity, purification, TAP), mass spectrometry (mass spectrometry), protein chip (protein chip) and based on Bioinformatics (Bioinformatics) analysis method. The bioinformatics analysis without specific experimental operation, is often studied in the whole genome scale, can obtain a large amount of information. According to the complementary protein crystal structure, can speculate on the possible interactions between proteins. Therefore, bioinformatics analysis provides a new direction for the research methods of protein interaction, provides an important clue for the experiment.
Our laboratory use differential gel electrophoresis (differential in-gel electrophoresis, DIGE) and mass spectrometry identified 22 mitochondrial content of endotoxic shock mice change within the protein, and the use of bioinformatics techniques for protein interaction prediction, the results found a group of protein interacting protein complexes: sterol carrier protein 2 (sterol carrier 2 protein, SCP2) / phytanic acid oxidase (phytanoyl-CoA, hydroxylase, Phyh). It is found in the experiment of lipopolysaccharide (lipopolysaccharide, LPS) to stimulate the mice after 1 hr of mitochondrial SCP2 and Phyh content reduced at the same time, and therefore, can reduce the ratio, it is speculated that they are both or with other proteins together with the function of the complex form through a channel through a mitochondria, which are involved in stress response to endotoxin. In addition, through We found that the analysis of these two proteins, both have some common characteristics, such as both in mitochondria localization, which are involved in fatty acid metabolism. On the basis of the above, we establish this study, using the related experimental methods to verify the interaction between SCP2/Phyh on mouse mitochondrial proteins. This experiment proved that if these two proteins are the existence of interaction, you can prompt the two of us is involved in fatty acid metabolism in complex form, which can provide a new direction for us to study some metabolic diseases associated with these two proteins.
Phyh protein was mainly localized in peroxisomes and mitochondria. The first reaction which is responsible for the catalytic oxidation of the peroxisomal alpha pathway, is phytane acyl coenzyme A into 2- hydroxy alkanoic acid and previous studies of coenzyme A. found that when the Phyh gene is mutated, can cause phytanic acid oxidation disorders, which led to a series of symptoms of the body the kidney and nervous system. The current research on the protein concentrates in Phyh gene mutation and Refsum's disease relationship. So far, foreign countries have been reported in several Phyh gene mutations, such as Arg275Trp, namely 275th arginine is tryptophan substitution. The mutant Phyh lost oxidation phytanic acid activity, resulting in a large number of phytanic acid metabolism, phytanic acid accumulation in various tissues and body fluids, resulting in Refsum's disease (Refsum's disease), cerebrohepatorenal syndrome (ceribro-hepato- Renal syndrome), proximal limb punctate cartilage dysplasia (rhizomelic chondrodysplasi) and neonatal adrenoleucodystrophy (neonatal adrenoleukodystrophy, NALD) and other peroxisome metabolic disorders related to. In addition, there are studies found that Phyh proteins can interact with some protein, resulting in Refsum's disease, such as brain tissue the interaction between PAHX-AP#1 protein and Phyh protein, and has some relation with Refsum's disease of the central nervous system symptoms.
SCP2 mainly exists in the mitochondria, peroxisomes and endoplasmic reticulum in the cytoplasm of the liver was the highest. On the one hand, as a regulator of cholesterol metabolism, biosynthesis and cholesterol in cholesterol to bile acids, cholesterol ester conversion and steroids; on the other hand as a cholesterol transporter, involved in intracellular membrane cholesterol transport, and the newly synthesized cholesterol liver directly from the endoplasmic reticulum to the rapid transport of bile. A large number of studies show that the increase of expression of SCP2 is an important cause of cholesterol gallstone disease.
In order to verify whether both Phyh and SCP2 interact with each other, we from the in vivo and in vitro respectively to verify the interaction between the two. First, we constructed Phyh prokaryotic and eukaryotic expression plasmids, respectively pGEX-4T-Phyh and pcDNA3-HA-Phyh SCP2; prokaryotic and eukaryotic expression plasmid, followed by pET14b-SCP2 and pcDNA3-Flag-SCP2. respectively, in order to Study on whether there exists interaction between them in vitro environment, we performed in vitro binding assays, we found that the two can bind in vitro. Then, we again from the body is verified. First using immunofluorescence technique showed two protein in the mitochondrial expression and co localization. Results showed that both of them can locate in the mitochondria, which were colocalized. Then using immunoprecipitation technique to observe both the in vivo results, and failed to detect both under normal conditions Binding in NIH/3T3 cells. So the idea of two only in certain stress to interact, therefore, in order to obtain the exact results, we further improved the method and experimental condition, was added respectively to stimulate NIH/3T3 cells 1H the two experimental group with NaAsO2 and LPS, the results have not yet been detected by the combination of the two. Analyzing the results, we believe this is due to the intracellular environment is more complex, both Phyh and SCP2 only in some special cases, in other intracellular substance will interact; or the interaction between the two is very weak and short-lived, the current experiment is difficult to verify whether the existence of the two. Interaction remains to be studied further.
In summary, this study in order to verify the interaction between Phyh and SCP2, first constructed two prokaryotic and eukaryotic expression plasmid, after experimental verification of both the in vitro interactions with in vitro, and two proteins in the cells of the co localization were observed by immunofluorescence, the immune co precipitation experiments to verify these interactions in vivo.
Through the above research, we get the following conclusions:
1. in vitro binding assay showed that Phyh and SCP2 could be specifically combined in the environment in vitro.
The immunofluorescence of 2. cells found that Phyh and SCP2 were Co located in the mitochondria.
3. the immunoprecipitation experiment shows that Phyh and SCP2 can not be specifically combined in the body, and the interaction of the two in the body needs further study.
The results of this study enrich the information of protein interaction and provide new ideas and methods for the study of new protein interaction.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【共引文献】