Analysis Of Pathological Mechanism Of Intermediate Filament Protein Vimentin Mutation E151K

Vimentin is a highly conserved type III intermediate filament protein.Intermediate filament is a major component of cytoskeleton, working together with microtubules to support and maintain the normal positioning of cellular organelles, correct connection between cells and proper adhesion, as well as migration of vertebrate cells.Genetic cataract is one of the major causes for blindness. Mutations in the genes coding for structure proteins, transcription factors, membrane proteins and ion channels all cause genetic cataract. It has been shown that the point mutation G596 A in the vimentin gene can cause cataract.Mechanistically, the G596 A mutation generates a protein with E151 K nonsense change, causing a loss of vimentin assembly and leading to cataract formation. However, why E151 K vimentin loses polymerization ability remains unknown.SUMOs are small Ubiquitin-like molecules. It can be linked to other proteins with the help of E1, E2 and E3 ligases. This process is called sumoylation, which plays an important role in cellular physiology including protein-protein interaction, localization of subcellular organelles,protein binding of DNA and regulation of enzyme activities.Our laboratory has conducted comprehension research on sumoylation control of eye development, especially lens development in the past decade. Our results have demonstrated that sumo1-mediated sumoylationis necessary to activate p32 Pax6 and thus regulate its downstream target genes to control eye development. In addition, our results have shown that sumo1-mediated sp1 conjiugation enhances its transcription activity and promotes cell differentiation. In contrast, sumo2/3-mediated sp1 sumoylation suppresses its function and inhibits lens differentiation.Since the E151 K mutation generates a conserved sumoylation site, we speculate that E151 K vimentin may be sumoylated and thus become incapable of assembling into functional intermediate filament, leading to abnormal functions of cytoskeleton.In this thesis, our team member Zhou Lu successfully constructed pCI-neo-vimentin expression vector and pCI-neo-E151K-vimentin expression vector, and established wild-type and mutant E151 K stable transformed cell lines. On the basis of her work, as two sumoylation sites,K313 and K373 are present in WT vimentin, I successfully constructed p CI-neo-K313R-vimentin site mutant expression vector,pCI-neo-K373R-vimentin site mutant expression vector,pCI-neo-K313R/K373R-vimentin double mutant expression vector, and pCI-neo-K313R/K373R/E151K-vimentin triple mutant expression vector.Using Lipofectamine 2000-mediated transfection, we introduced pCI-neo-K313R/K373R-vimentin,pCI-neo-K313R/K373R/E151K-vimentin and pCI-neo vector into α-TN4-1 cells. Under G418 screening, we obtained the stable clonesexpressing either double mutant or triple mutant vimentin, which were further confirmed by western blot and immunocytochemistry. Through comparison of vimentin expression, sumoylation status and cellular morphology in wild type, E151 K, K313R/K373 R, and E151K/K313R/K373 R mutant transfected cells, we came to the conclusion that the mutant vimentin is likely sumoylated at E151 K site causing the loss of assembly, and leading to cataract formation. Together, our results for the first time reveal the molecular mechanism of cataract formation derived from E151 K mutation in vimentin.