Research On Screening And Identification Of Proteins Interacting With Mutant SOD1 From A FALS

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.