本文關(guān)鍵詞： 蛋白質(zhì)相互作用 植烷酸氧化酶 固醇攜帶蛋白　出處：《南方醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：生物體內(nèi)生命攜帶的遺傳信息由不同的基因經(jīng)轉(zhuǎn)錄、翻譯傳遞到相應(yīng)的蛋白質(zhì)上并使其具有各自的生化特性及生物學(xué)活性；但每個蛋白質(zhì)并不是獨立地在細胞中完成被賦予的功能,它們在細胞中通常與其他蛋白質(zhì)相互作用形成大的復(fù)合體,在特定的時間和空間內(nèi)完成特定的功能,而且有些蛋白質(zhì)的功能只有在復(fù)合體形成后才能發(fā)揮出來。蛋白質(zhì)間的相互作用可改變細胞內(nèi)蛋白質(zhì)的動力學(xué)特征,如底物結(jié)合特性、催化活性；也可產(chǎn)生新的結(jié)合位點,改變蛋白質(zhì)對底物的特異性；還可使其它蛋白質(zhì)失活,調(diào)控其它基因表達。只有蛋白質(zhì)間相互作用順利進行,細胞的正常生命活動過程才有保障。因此,研究蛋白質(zhì)相互作用,對于深入了解蛋白質(zhì)的功能,闡明生命活動的規(guī)律有著重要的意義。 目前用于研究蛋白質(zhì)間相互作用的方法包括酵母雙雜交(yeast-two hybrid)、體外結(jié)合實驗(in vitro binding)、免疫共沉淀(coimmunoprecipitation)、串聯(lián)親和純化(tandem affinity purification, TAP)、質(zhì)譜鑒定(mass spectrometry)、蛋白芯片(protein chip)以及基于生物信息學(xué)(Bioinformatics)的分析方法等。其中,生物信息學(xué)分析無需具體的實驗操作,往往是在整個基因組規(guī)模上進行研究,可獲得較大的信息量。另外根據(jù)蛋白晶體結(jié)構(gòu)的互補性,也可以推測蛋白間可能的相互作用。因此,生物信息學(xué)分析為研究蛋白質(zhì)相互作用的方法提供了一個新的方向,為實驗提供了重要的線索。 本實驗室前期利用差異凝膠電泳(differential in-gel electrophoresis, DIGE)和質(zhì)譜分析技術(shù)鑒定出內(nèi)毒素休克小鼠線粒體內(nèi)22種含量發(fā)生改變的蛋白質(zhì),并運用生物信息學(xué)技術(shù)進行了蛋白質(zhì)相互作用預(yù)測,結(jié)果找到了一組具有相互作用的蛋白-蛋白復(fù)合物：固醇攜帶蛋白2(sterol carrier protein 2, SCP2)／植烷酸氧化酶(phytanoyl-CoA hydroxylase, Phyh)。實驗發(fā)現(xiàn)在脂多糖(lipopolysaccharide, LPS)刺激小鼠1 hr后線粒體內(nèi)SCP2和Phyh的含量同時減少,并且減少倍數(shù)一致,因此,可以推測它們可能是兩者或與其他蛋白一起以功能復(fù)合物的形式通過某種通道穿梭出線粒體,從而參與機體對內(nèi)毒素的應(yīng)激反應(yīng)。另外,通過對這兩個蛋白進行分析我們發(fā)現(xiàn),兩者存在一些共性,例如兩者在線粒體中均有定位,且都有參與脂肪酸代謝。在上述基礎(chǔ)上,我們確立了此項研究,即運用相關(guān)實驗方法來驗證SCP2/Phyh這對小鼠線粒體蛋白質(zhì)之間的相互作用。如果實驗證明這兩個蛋白確實存在相互作用,就可以提示我們兩者是以復(fù)合物的形式參與脂肪酸代謝,這可以為我們研究與這兩個蛋白相關(guān)的一些代謝性疾病提供新的方向。 Phyh蛋白主要定位于過氧化物酶體和線粒體中。它負責(zé)催化過氧化物酶體α-氧化途徑的首個反應(yīng),即將植烷酰輔酶A轉(zhuǎn)化為2-羥基植烷酸輔酶A。先前的研究發(fā)現(xiàn),當(dāng)Phyh基因發(fā)生突變時,可引起植烷酸氧化障礙,進而導(dǎo)致機體腎臟和神經(jīng)系統(tǒng)的一系列癥狀的產(chǎn)生。目前對該蛋白的研究多集中在Phyh基因的突變與雷弗素姆病的關(guān)系上。迄今為止,國外已經(jīng)陸續(xù)報道了幾種Phyh基因的突變,例如Arg275Trp,即第275位的精氨酸被色氨酸取代。突變的Phyh失去氧化植烷酸的活性,進而導(dǎo)致植烷酸代謝障礙,大量植烷酸蓄積于各種組織和體液中,最終引起雷弗素姆病(Refsum's disease)、腦肝腎綜合征(ceribro-hepato-renal syndrome)、肢近端型斑點狀軟骨發(fā)育不良(rhizomelic chondrodysplasi)和新生兒腎上腺白質(zhì)營養(yǎng)不良癥(neonatal adrenoleukodystrophy, NALD)等過氧化物酶體相關(guān)性代謝性疾病。此外,還有研究發(fā)現(xiàn)Phyh蛋白可以和一些蛋白發(fā)生相互作用,進而導(dǎo)致雷弗素姆病的發(fā)生,例如腦組織中的PAHX-AP#1蛋白可與Phyh蛋白發(fā)生相互作用,并與雷弗素姆病中樞神經(jīng)系統(tǒng)方面的癥狀有一定的聯(lián)系。 SCP2主要存在于線粒體、過氧化物酶體、內(nèi)質(zhì)網(wǎng)和胞質(zhì)中,肝臟含量最高。它一方面作為膽固醇代謝的調(diào)節(jié)因子,參與膽固醇的生物合成和膽固醇向膽汁酸、膽固醇酯及類固醇激素的轉(zhuǎn)化；另一方面作為膽固醇轉(zhuǎn)運器,參與細胞內(nèi)、質(zhì)膜間膽固醇的運輸,并能將肝臟新合成的膽固醇直接從內(nèi)質(zhì)網(wǎng)快速轉(zhuǎn)運至膽汁。大量研究表明SCP2表達水平的升高是膽固醇結(jié)石發(fā)生的重要原因。 為了驗證Phyh和SCP2兩者是否具有相互作用,我們從體內(nèi)和體外分別驗證了兩者的相互作用。首先,我們構(gòu)建了Phyh的原核和真核表達質(zhì)粒,分別為pGEX-4T-Phyh和pcDNA3-HA-Phyh;SCP2的原核和真核表達質(zhì)粒,分別為pET14b-SCP2和pcDNA3-Flag-SCP2。緊接著,為了研究兩者在體外環(huán)境下是否存在相互作用,我們進行了體外結(jié)合實驗,結(jié)果發(fā)現(xiàn)兩者在體外可以特異性地結(jié)合。然后,我們又從體內(nèi)進行了驗證。先利用免疫熒光技術(shù)觀察兩個蛋白在線粒體的表達和共定位情況。結(jié)果發(fā)現(xiàn)兩者均能定位于線粒體中,且存在共定位。而后運用免疫共沉淀技術(shù)觀察兩者的體內(nèi)結(jié)合情況,結(jié)果在正常情況下并未能檢測到兩者在NIH/3T3細胞中的結(jié)合。于是設(shè)想兩者僅在某些應(yīng)激刺激下才發(fā)生相互作用,因此,為了獲得確切的實驗結(jié)果,我們進一步改進了實驗方法和條件,增設(shè)了分別用NaAsO2和LPS刺激NIH/3T3細胞1h這兩個實驗組,結(jié)果仍未檢測到兩者的結(jié)合。對此結(jié)果進行分析,我們認(rèn)為可能是由于細胞內(nèi)環(huán)境比較復(fù)雜,Phyh和SCP2兩者只有在某種特殊情況下,在胞內(nèi)其他物質(zhì)的作用下才會發(fā)生相互作用；或者是兩者的相互作用很弱且短暫,通過當(dāng)前的實驗難以驗證。究竟兩者是否存在這種相互作用,還有待更深入的研究。 綜上所述,本研究為了驗證Phyh和SCP2之間是否存在相互作用,首先構(gòu)建了兩者的原核和真核表達質(zhì)粒,之后通過體外結(jié)合實驗驗證了兩者在體外的相互作用,然后通過細胞免疫熒光技術(shù)觀察兩個蛋白在細胞內(nèi)的共定位情況,最后通過免疫共沉淀實驗來驗證兩者在體內(nèi)的相互作用。 通過以上研究,我們得到以下結(jié)論： 1.體外結(jié)合實驗表明Phyh和SCP2在體外環(huán)境中能特異性地結(jié)合。 2.細胞免疫熒光發(fā)現(xiàn)Phyh和SCP2在線粒體中有共定位。 3.免疫共沉淀實驗表明Phyh和SCP2在體內(nèi)不能特異性地結(jié)合,兩者是否在體內(nèi)具有相互作用還需進行進一步的研究。 本課題的研究結(jié)果豐富了蛋白質(zhì)相互作用的信息,并為進行新的蛋白質(zhì)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

【共引文獻】

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1 樊晉宇;基于mCherry的雙分子熒光互補系統(tǒng)研究[D];華中科技大學(xué);2008年

2 錢冠華;NIRF蛋白對HBV核心蛋白的作用及其機制的初步研究[D];重慶醫(yī)科大學(xué);2012年

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1 劉震西;PRDX1與PRAK相互作用研究[D];東北林業(yè)大學(xué);2012年

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