【作者】 陈桂生；

【导师】 李露斯；

【作者基本信息】 第三军医大学 ， 神经病学， 2008， 博士

【摘要】 背景肌萎缩侧索硬化症(amyotrophic lateral sclerosis, ALS)是较常见的成年发病、进行性发展的、致死性的神经元变性疾病,损伤大脑皮质、脑干、脊髓运动神经元。临床治疗非常困难,患病后3-5年因呼吸衰竭死亡。ALS病理特点为上运动神经元(锥体束)和下运动神经元(脊髓前角细胞及脑干运动核)均出现不同程度的变性,临床表现为脊髓和脑干所支配的肌肉无力、萎缩、肌束震颤、球麻痹和锥体束征。ALS可分为家族性肌萎缩侧索硬化症(familial amyotrophic lateral sclerosis FALS)和散发性肌萎缩侧索硬化症(sporadic amyotrophic lateral sclerosis SALS)。大多数ALS病例为SALS,占80%-90%, FALS占10%～20%,多数表现为常染色体显性遗传。Siddique等以微卫星DNA标记对6个FALS家系进行遗传连锁分析,将FALS基因定位于21号染色体长臂。已确认此区主要包括铜锌超氧化物歧化酶(Cu/ZnSOD1)、谷氨酸受体亚单位GluRS、甘氨酰胺核苷酸甲酰转移酶催化酶基因,现在认为FALS的发病与超氧化物歧化酶1(superoxide distmase 1 ,SOD1)基因突变关系密切,约20%FALS是由于SOD1基因突变所致。转基因鼠的培育成功更进一步证明了SOD1基因突变为FALS的致病基因。目前SOD1基因突变类型在FALS中多达119种。人们希望通过对突变SOD1(mutation SOD1,mSOD1)基因的研究,以揭示mSOD1的生理功能,阐明ALS的病理机制。蛋白质-蛋白质的相互作用是细胞生命活动的基础和特征,也是疾病发生发展的病理生理的重要过程。研究生命活动过程中蛋白质间相互作用有助于揭示生命过程的许多本质问题。酵母双杂交系统是近年来发展起来的一种分析真核细胞中蛋白质-蛋白质相互作用的有效手段,为研究蛋白质在体内生理情况下的相互作用提供了一种新的遗传学方法。酵母双杂交系统通过将两个假定有相互作用的蛋白X和Y分别融合到一酵母转录激活因子的BD和AD上,X与Y的相互作用重构了转录激活因子从而导致下游报告基因的转录,产生容易探测到的表型。本实验选用的是Clontech公司的MATCHMAK-ER酵母双杂交系统3,单倍体细胞AH109株带有4种报告基因,分别为H1S(组氨酸)、ADE2(腺苷)、MEL1(半乳糖苷酶)和LACZ(半乳糖苷酶)。加上两种载体中分别带有的TRP1(色氨酸)和LEU2(亮氨酸),使得筛选后的阳性菌落能在缺乏上述4种氨基酸营养的培养板上生长。而且MEL1基因表达的半乳苷酶为分泌型蛋白。可以在固相培养基中直接与X-α-gal底物相互作用,显示为蓝色,相对于检测β半乳糖苷酶来说操作得到了极大的简化。该系统由于增加了报告基因的种类,使筛选结果阳性率达到95%。虽然酵母双杂交系统已成为生物学家研究蛋白相互作用的标准方法,但不能排除由于酵母生物本身特征而产生的非真实相互作用。所以在用酵母双杂交系统进行文库筛选之后,通过多种方法进行验证。如利用免疫共沉淀技术对其进行验证,进一步确定相互作用的存在。我科于1999年在重庆市潼南县发现的一个ALS家系具有与目前文献报道完全相同的临床表型。我们应用参数连锁分析的方法对该家系进行了初步研究,发现该家系致病相关基因与位于21号染色体上的微卫星标记D21S223位点相连锁。SOD1是位于21号染色体上D21S223位点附近唯一的特征性基因,因此我们推测,该家系发病的原因可能是由SOD1基因突变引起的,同时可能存在新的SOD1突变类型。我们采用聚合酶链-单链构象多态性分析(polymerase chain reaction single-strand conformation polymorphism,PCR-SSCP)法对该ALS家系SOD1基因进行了突变分析,结果发现,该家系部分成员SOD1基因2号外显子编码区和5号外显子非编码区发生了插入突变,这可能是新的SOD1基因突变类型,其中2号外显子的突变可能为该家系发病的原因。但是该突变的SOD1基因功能和编码蛋白质的作用还不清楚。蛋白质是基因功能的体现者和执行者,直接对蛋白质的表达模式和功能模式进行研究就成为生命科学发展的必然趋势。通常要对一个蛋白质进行深刻的了解,就必须得到下面几方面的信息:(1)哪一个区域是该蛋白质进化上的保守结构域;(2)该蛋白质的表达水平;(3)该蛋白质在细胞内的分布;(4)翻译后的修饰和加工;(5)哪些蛋白质与该蛋白质存在相互作用。鉴定一个新蛋白质的功能时,一个重要的方面是鉴定与其相互作用的蛋白质。文献检索,目前未见与mSOD1相互作用蛋白的研究报道。本实验采用Clontech公司的MATCHMAK-ER酵母双杂交系统3筛选与mSOD1相互作用的蛋白,以揭示mSOD1的功能。目的(1)确定该mSOD1是否为新的SOD1突变基因;寻找mSOD1的同源基因;明确mSOD1cDNA序列保守性和相似性,以确定mSOD1在进化过程中是否还保持野生型SOD1一样的功能;编码蛋白亚细胞定位。明确mSOD1的生物学特性的目的是为酵母双杂交系统筛选相互作用蛋白提供参考。(2)构建表达“诱饵”融合蛋白的质粒pGBKT7-mSOD1cDNA。重组质粒pGBKT7-mSOD1cDNA转化酵母AH109,明确表达“诱饵”融合蛋白质粒在酵母AH109中表达及在酵母AH109中自激活作用和毒性作用。(3)获取与mSOD1相互作用蛋白基因序列。(4)与mSOD1相互作用蛋白基因序列的同源性搜索,确定筛选的相互作用蛋白是已知蛋白还是未知蛋白。分析已知蛋白的功能。(5)验证酵母双杂交结果,确定mSOD1与相互作用蛋白的相互作用关系真实存在。方法(1)利用NCBI在线工具分析mSOD1:以人类基因组数据库为基础,利用BLAST程序分析mSOD1的基因结构和序列相似性搜索;基因的染色体定位和编码蛋白亚细胞定位;DNAstar软件分析ORF finder。(2)按照引物设计原则,按照pGBKT7载体序列和mSOD1cDNA序列设计引物,保证该基因的开放阅读框和翻译时不发生碱基突变,含EcoRⅠ/SalⅠ双酶切位点;应用PCR技术扩增mSOD1cDNA片断,对PCR产物EcoRⅠ/SalⅠ双酶切琼脂糖凝胶电泳和测序验证。(3)通过限制性内切酶EcoRⅠ/SalⅠ双酶切载体pGBKT7和mSOD1cDNA,用T4 DNA连接酶连接双酶切产物,转化感受态DH5α,筛选重组子,进行双酶切琼脂糖凝胶电泳和测序验证。(4)应用LiAC法,将重组质粒pGBKT7-mSOD1cDNA转化酵母AH109,应用PCR技术验证转化结果。检测重组质粒pGBKT7-mSOD1cDNA对酵母AH109的毒性作用和在酵母AH109中自激活作用。SDA-PAGE、Western蛋白印记检测重组质粒pGBKT7-mSOD1cDNA的融合蛋白在酵母AH109中的表达。(5)采用Clontech第三代的酵母杂交系统,利用酵母a型和α型单倍体细胞相互结合的原理,结合酵母营养缺陷培养基和酵母菌株携带的报告基因,筛选与mSOD1相互作用的蛋白。(6)利用生物信息学方法对筛选的相互作用蛋白质基因的氨基酸序列进行分析。(7)应用免疫共沉淀技术验证mSOD1与相互作用蛋白质在哺乳动物细胞内相互作用关系的真实存在。结果(1)确定mSOD1是一个新的突变SOD1基因, GenBank ID:EF143990。mSOD1cDNA序列全长108bp,定位于染色体21q21.2,其开放阅读框为108bp,编码36个氨基酸,氨基酸序列具有高度的保守性,与其它突变类型的SOD1基因具有很高的同源性。(2)成功构建表达“诱饵”融合蛋白的质粒pGBKT7-mSOD1cDNA。(3)成功地将重组重组质粒pGBKT7-mSOD1cDNA转化酵母AH109;重组质粒pGBKT7-mSOD1cDNA的融合蛋白在酵母菌中有稳定的表达;重组质粒pGBKT7-mSOD1cDNA对酵母AH109无毒性作用,重组质粒pGBKT7-mSOD1cDNA的融合蛋白在酵母AH109中无自激活作用。(4)获得15个与mSOD1相互作用的蛋白质,8个已知蛋白,7个未知蛋白。8个已知蛋白为:蛋白酪氨酸磷酸酶,非受体2(PTPN2);TBC1D4;蛋白激酶族,富含精氨酸、丝氨酸剪切因子2;SRC家族酪氨酸激酶Fyn;营养障碍基因糖蛋白β-Sarcoglycan;甘氨酸受体α2;微管结合蛋白相关蛋白MAP/MARK1;铁蛋白重链。(5)证实mSOD1与MAP/MARK1和β-Sarcoglycan的相互作用真实存在。结论(1)这个ALS家系突变的SOD1是SOD1新的突变类型,是ALS的致病基因。(2)应用酵母双杂交系统成功地筛选出15个与mSOD1相互作用蛋白,8个已知蛋白,它们是蛋白酪氨酸磷酸酶,非受体2(PTPN2);TBC1D4;蛋白激酶族,富含精氨酸、丝氨酸剪切因子2;SRC家族酪氨酸激酶Fyn;营养障碍基因糖蛋白β-Sarcoglycan;甘氨酸受体α2;微管结合蛋白相关蛋白MAP/MARK1;铁蛋白重链。其余7个未知蛋白。证实mSOD1与MAP/MARK1和β-Sarcoglycan相互作用的存在。使mSOD1的功能研究又取得新的进展。(3) ALS的病理机制尚未明确,通过本实验结果推测,可能是mSOD1与之相互作用蛋白形成网络相互影响,在ALS发病中扮演了重要的角色,这可能是ALS发病的分子病理基础。本实验结果为ALS的病理机制研究提供了新的依据和线索。(4)生物信息学是进行生物医学实验不可缺少的平台,目前在生物医学的各个领域起到越来越重要作用。生物信息学分析为本实验筛选与mSOD1相互作用的蛋白分子提供了良好的参考。(5)酵母双杂交系统是筛选相互作用蛋白的高效、灵敏的方法。更多还原

【Abstract】 BackgroundAmyotrophic lateral sclerosis(ALS) is the most common adult onset neurodegenerative disorder characterized by the degeneration of large motor neurons in the cerebral cortex,brain stem and spinal cord .Dysfunction and death of these cell populations lead to progressive amyasthenia and atrophy.The clinical therapy is very difficult,and ultimately ,patients will be paralysis and die within 3 to 5 years after disease onset,typically from respiratoy failure. Pathological character of ALS is distinct level degenerative disorder for upper motor neurons (tractus pyramidalis) and lower motoneurons (anterior horn cell of spine,motor of brain stem),and its clinical manifestation is muscle weakness ,atrophy , fasciculation , glossopharyngeal paralysis and pyramid sign. ALS is divided into familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS),of which 80%～90% are SALS cases,of which 10%～20% are FALS cases,and most of the FALS cases have autosomal dominant traits. Siddique et al reported that was genetic linkage analysis carried out by microsatellite marker for sixes FALS, and gene-mapping of FALS localizated on chromosome 21 long arm,where it is the affirmated that there are three kinds of kinase gene in the domain, including Cu/Zn superoxide dismutase (SOD1),GluR5,glycinamide ribonucleotide formylacyl transferase . At present, it is believed that the onset of FALS has close relations with the mutation SOD1 gene , and 25% of the FALS cases were reported to have a mutation in the Cu/ZnSOD1 gene. And trangenic mice experimentation evidenced that virulence gene of FALS is mutation SOD1 gene.Up to now, there are 119 kinds mutation types of SOD1 found in ALS.People want to study the encoding protein of mutation SOD1 in order to know the pathological mechanisms of ALS.Proteins rarely work alone themselves. They almost always interact with other biomolecules to execute their functions. Protein-protein interactions (or PPIs) are key elements for the normal functioning of a living cell. Knowledge of specific protein–protein interactions is an important component in understanding biological processes and disease mechanisms.The yeast two-hybrid (Y2H) system is a especially useful method to analyse interactions of protein - protien in vivo, and a new genetics assay for detecting protein-protien interactions in vivo. The Y2H system was on the basis of modular domain structure of the transcription factor GAL4, comprised of a DNA binding domain and transcription activation domain. In the Y2H system, one of the proteins of one’s interest, termed X, is expressed as a hybrid protein with the GAL4 DNA binding domain, whereas the other, termed Y, is expressed with the activation domain. If X and Y interact, the two hybrid proteins, often coined as“bait”and“prey”, respectively, are assembled onto GAL4 binding sites in the yeast genome. The assembly functionally reconstitutes the GAL4 transcription factor and induces the expression of reporter genes integrated in the region downstream of the GAL4 binding sites.We used MATCHMAK-ER yeast two-hybrid system 3 of Clontech company, haploid AH109 strain has four kinds report gene:HIS,ADE2,MEL1 and LacZ.In addition to two vectors to part TRP1 and LEU2,screening positive colony can culture to lack four amino acids,theα-Gal quantitative assay can be used to measure the extracellularα-galactosidase activity produced during the culture of any yeast strain that carries the MEL1 gene, MEL1 is endogenous to many but not all yeast strains,opposite to detectβ- galactosidase, this handling is easy. The system increases type of report gene ,and positive ratio of screening is 95%.However,yeast two-hybrid system is a standard method for biologists to study protein inter-actions ,it cannot exclude false protein-protein interactions because of biological feature of yeast cell itself. So it is necessary to identi-fy the results form the yeast two-hybrid system.For instance, co-immun-oprecipitation is a common method which used to determine protein–protein interactions.There is not any report of the genetic research of FALS on mutant SOD1 in China . However, we found a family ALS in Tongnan County, Chongqing, in 1999, in which nosogenic members had a similar clinical phenotype of the disease, this family is inherited as an autosomal dominant trait.We performed genetic linkage analysis with D21S223 marker and found that this FALS is linked to chromosome 21q microsatellite marker. As SOD1 gene is the only one characteristic gene nearby D21S223 marker, we supposed that this FALS is a new type of ALS caused by a new mutation in SOD1, which is different from what have been reported. PCR-SSCP analysis and sequencing found that a base pair insertion mutation occurred in ecoding region of exon 2 and noncoding region of exon 5. This is a new type of SOD1 mutations, which may be responsible for the disease of this family. The common informations understand concerning protein :(1)Where is conservative structural domain of the protein evolution?(2)the proteinaceous express level;(3)where is intracellular distribution of the protein?(4) modification and process about post-translation; (5) interaction between the protein and other proteins. We will research these problems of encoding protein on mutation SOD1.Through literature retrieval,at present,there is not any report of protein–protein interactions with mSOD1.We will screen proteins that interact with mSOD1 from the human fetal brain cDNA library using MATCHMAKER yeast two-hybrid system 3 ,and to reveal the function of mSOD1.Purposes(1) Mutation SOD1cDNA was analyzed by bioinformatics to determine whether this mutation gene is a novel mutation; we want to know whether or not homologization with others mutation SOD1 in ALS; we want to know its conservation,and subcellurlar localization,and its gene expression map.We predicted the function of mSOD1.(2) The fragment of mSOD1cDNA was subcloned into pGBKT7 vector of yeast eukaryotic expression to construction bait vector.Then,recombinant plasmid pGBKT7- mSOD1-cDNA was transformed into AH109.(3) We screened the human fetal brain cDNA library to find proteins that interact with mSOD1.(4) The sequence of positive clone was analyzed by bioinformatics, we align its homology, we want to know interactive proteins whether know proteins or unknown proteins.(5) We confirmed results from the yeast two-hybrid system, whether real interaction between mSOD1 and interactive proteins.Methods(1) With online tool BLASTn of NCBI to analysis of mSOD1. Based on the human genome resource, the gene sequence of mSOD1 was analyzed by DNAstar tool and BLAST procedure, we searched the similar gene with mSOD1; Where it is the chromosome location , and tissue expression, and spatial structure of protein . (2) The fragment of mSOD1cDNA was amplified from pEGFP-N2mSOD1cDNA by polymerase chain reaction( PCR).Then,the fragments were identified by SalⅠand EcoRⅠand sequencing analysis.(3) The fragment of mSOD1cDNA was subcoloned into pGBKT7 vector by action of T4 ligase. Recons of pGBKT7-mSOD1cDNA were transformed competence E.coli DH5α,recons of pGBKT7-mSOD1cDNA were identified by double enzyme cutting and sequencing analysis.We obtained the correct recombinant plasmid pGBKT7-mSOD1cDNA from E.coli DH5α.(4) Recombinant plasmid pGBKT7-mSOD1cDNA was transformed into the competent AH109 by LiAC and identified using PCR. The self-activation of pGBKT7-mSOD1cDNA on the reported genes and its toxicity on AH109 were also determined,the expression products of fusion protein were identified by SDS-PAGE and Western blot.(5) We screened the human fetal brain cDNA library to find proteins that interact with mSOD1 using MATCHMARKER yeast two-hybrid system 3,the principle is mating between a type yeast strains andαtype yeast strains.Then, between bait protein and plasmid protein of cDNA library interacted in diploid cell and activated gene expression.We obtained positive clones .We analyzed the sequence of positive clone by bioinformatics.(6) We confirmed interactions between mSOD1 and interactive proteins with the method of co-immunoprecipitation in vitro.Results(1) Mutant SOD1 was analyzed by bioinformatics:Mutation SOD1 is a novel mutation gene ,GenBank ID is EF143990, the full length of the mutant SOD1 is 108bp,which is located in 21q21.2,and the ORF was 108bp.The similar search shows the highest homology with human sapiens mutation SOD1 gene from ALS .(2) The mSOD1cDNA fragment was successfully subcoloned into pGBKT7 vector.We obtained recombinant plasmid pGBKT7-mSOD1cDNA.(3) The recombinant plasmid pGBKT7-mSOD1cDNA was correctly transformed into yeast AH109, there were no self-activation of pGBKT7-mSOD1cDNA on the reported genes and no toxicity on AH109,the fusion proteins could expression in AH109.(4) Using the Y2H system ,we obtained 15 positive clones ,of them are 8 known proteins,the other are 7unknown proteins.known proteins are PTPN2, TBC1D4, SFRS2, FYN,GlyRα2,MAP/MARK1,β-Sarcoglycan,Ferritin H.(5) Results of the yeast two-hybrid system were confirmed that are real interactions between mutation SOD1 and interactive proteins by co-immunoprecipitation technique.Conclusion(1) The mutant SOD1 is a novel mutant type of SOD1, which may be responsible for the disease of this family.(2) We obtained 15 interactive proteins with mutant SOD1 by the yeast two-hybrid system, of which are 8 know proteins,they are PTPN2, TBC1D4,SFRS2,FYN, GlyRα2, MAP/MARK1,β-Sarcoglycan and Ferritin H.The other are unknown proteins. We confirmed protein-protien interactions between mutant SOD1 and MAP/MARK1 orβ-Sarcoglycan.(3) We knowned the function of mSOD1 in understanding the function of known proteins. It is an important component in understanding molecular pathological mechanisms of ALS,(4) Bioinformatics is playing a more and more important role in biomedical research regions.(5) The yeast two-hybrid (Y2H) system is a especially useful method to analyse interactions of protein - protien in vivo, and a new genetics assay for detecting protein-protien interactions in vivo.更多还原