Screening New Binding-partners Of Nogo-66 By Yeast Two-hybrid System

Axonal regeneration may be inhibited by CNS myelin and neuro-glial scar that arises from the lesioned tissue dramatically. Among which several proteins have been identified as components that can inhibit outgrowth of axon. Myelin-Associatd Glycoprotein(MAG), Oligodendrocyte Myelin Glyco-protein (Omgp) and Nogo are all proteins with significant neurite outgrowth inhibitory activity. Study on neurite outgrowth inhibitors may be therapeutically beneficial for patients with nerve injury or some neurodegenerative diseases such as Alzheimer's and Parkinson's disease. However, the mechanism responsible for Nogo-induced growth inhibition remains elusive.In present study, the human brain cDNA library was screened using yeast two-Hybrid system, and several new intracellular binding-partners of Nogo-66 protein have been identified. Necdin and GAPDH were confirmed to interact with Nogo-A using the method of immunoprecipitation in Vivo and Vitro. Both Necdin and GAPDH are highly co-localized with Nogo-A in cell body and neurite. Nogo-A retained Necdin in cytoplasmic region of HEK293 cells. Overexpression of Nogo-A attentuated accelerated effect induced by Necdin in PC12 cells.All together, the adult human brain cDNA library were screened with Nogo-66 as bait using yeast two-hybrid and several candidates were obtained. The physical association between Necdin or GAPDH with Nogo-A were further confirmed. Meanwhile, the protein-protein interaction between Nogo-A and Necdin were detected in yeast. Our data indicated that Nogo-A may retain Necdin in cytoplasma and attentuated accelerated differentiation induced by Necdin in PC12 cells. The above results have not been reported.