Screening Of LRRK2, A Gene Related To Parkinson's Diease, Interactants By Yeast 2-hybrid Analysis

Parkinson's disease(PD),a disease characterized by tremor,rigidity, bradycinesia and postural instability,is a chronic and progressive neurodegenerative disorder of the central nervous system that impairs control of voluntary movement.While the majority of cases are idiopathic,～5-15%of cases have a genetic component.Studying these rare genetic forms of PD has highlighted key cellular pathways,which are believed to also be involved in the pathogenesis of the more common sporadic forms of disease.The pathological hallmarks of PD patients include progressive loss of dopaminergic(DA)neurons in the substantia nigra pars compacta as well as the presence of ubiquitin-positive Lewy neuritis and Lewy bodies in the remaining neurons,α-synuclein,Parkin,UCH-L1,PINK1,DJ-1,LRRK2, ATP13A2 and HtrA2 are linked to inherited form of PD.LRRK2 gene has 51 exons.The LRRK2 protein has 2527aa,including ANK,LRR,ROC,COR,Kinase,WD40 six domains.The discovered mutations associated with PD are distributed in all the domains of LRRK2 and the most common one is G2019S,The MW of LRRK2 is 280 KD.It is expressed in brain,other organs and cell line HEK293T with unclear functions. Some research have showed that mutations associated with PD,such as R1441C,G2019S,I2020T,lead to increasing of LRRK2 kinase activity and decreasing of cell activity,indicating that these mutations are toxic to cells. Moreover,LRRK2 plays a role in neurite process morphology and polarity of synaptic vesicles(SV)protein.Employing a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait,we performed a yeast two-hybrid analysis to isolate and identify its potential binding partners in the human fetal brain cDNA library.The bait was amplified by polymerase chain reaction(PCR)and the fragment was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-Bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-Bait in AH109 yeast strain.Then human fetal brain cDNA library was transformed into that yeast strain,which could express pGBKT7-Bait fusion protein.Therefore the yeast strain which contained pGBKT7-Bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-α-gal.We retested these positive colonies by using of two independent yeast strains AH109 contained pGBKT7-Bait or pGBKT7 respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Finally,we obtained 9 true positive colonies and these colonies were then sequenced and performed blast in GeneBank.3 colonies of the 9 positive colonies were not in open reading- frames.Among other 6 colonies,there are known proteins including STRBP(spermatid perinuclear RNA-binding protein)and BAG5(BCL2-associated athanogene 5 isoform b),as well as unknown proteins including PTPN23(tyrosine phosphatase non-receptor type), L3MBTL3(1(3)mbt-like 3 isoform b),RALYL(RALY RNA binding protein-like isoform 1)and KIAA1783(Homo sapiens mRNA for KIAA1783 protein,partial cds).True positive colonies were successfully obtained from the yeast two-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,PD pathogenesis and other neurodegerations.