Numb Protein Modulates The Mono-ubiquitination Of α-synuclein Protein

BackgroundParkinson’s disease (PD) is a common neurodegenerative disease characterizedby bradykinesia, rigidity and resting tremor. In the brain of Parkinson’s patient,neurons death are mostly located in the substantia nigra region of the brain, and moredeaths before clinical symptoms appear, about70%of the dopamine neurons mayhave died when clinical symptoms become apparent. Pathological characteristics ofParkinson’s disease is the inclusion formation of α-synuclein (α-syn) protein, theprotein is the main component of Lewy bodies. Accumulation of abnormally foldedα-syn proteins form oligomers, the cytotoxicity may be a major pathologicalprocesses in the pathogenesis of Parkinson ’s disease.Numb, an evolutionarily conserved adaptor protein, is necessary for the cellfate specification of progenitor cells in the Drosophila nervous system.The Numbprotein has multiple protein-protein interaction regions including a phosphotyrosinebinding (PTB) domain and a carboxy-terminal domain that contains conservedinteraction motifs including an EH domain binding motif andα-adaptin binding site.By contacting with ubiquitin ligase, such as Siah1, Lnx and other interactions, itcontributes to the target protein ubiquitination. Numb also has the function inendosomal recycling and intracellular trafficking of receptors. In addition, Numb is an endocytic protein involved in neurite outgrowth and cell migration. Also Numband Notch protein through mutual antagonism play a role of multiple biochemicalsignaling pathways involved in the regulation, regulating adhesion and tightconnections in many processes. Studies have shown that Numb/Notch signalingpathway regulated the ubiquitination of some protein by the ubiquitin-proteasomesystem.There have no been reported that Numb protein regulates themono-ubiquitination level of α-syn protein at home and abroad, This paper aim toexplore whether Numb protein by taking part in the regulation of α-synmono-ubiquitination level plays a role in the pathogenesis of Parkinson’s disease, andto provide new ideas and methods for early diagnosis and treatment of Parkinson’sdisease.ObjectiveTo investigate the regulation of Numb on the level of α-synuclein mono-ubiquitination.MethodsTransfecting the recombinant plasmid EGFP-N1, EGFP-Numb, HA-α-synucleininto SH-SY5Y cells with the eukaryotic cell transfection technique. After transfected,we examined colocalization between α-synuclein and Numb by immunofluorescenceconfocal microscopy. And then, we performated co-immmunoprocipitation toexamine theinteraction between α-synuclein and Numb. Futhermore, by Western-blotto examine the effect of Numb overexpression on the level of α-synucleinubiquitination. Immunostaining to observe the formation of α-synuclein-positiveinclusion bodies in the SH-SY5Ycells treated with1mmol/L MPP+.ResultsIn the overexpression EGFP-Numb group, the Numb showed a significant overlap with α-synuclein, indicating the Numb colocalizes with α-synuclein at asubpopulation in cells compared to the control group; SH-SY5Y cells co-transfectedwith EGFP-Numb and HA-α-syn were treated with10μM MG132, the lysates wereimmunoprecipitated and detected by Western-blot, revealling that overexpressionNumb increases the level of mono-ubiquitinated α-synuclein; Immunostaining ofSH-SY5Ycells treated by1mmol/L MPP+with anti-α-syn antibody, we fundα-synuclein aggregates accumulate in the perinuclear area.ConclusionNumb increases the level of mono-ubiquitination α-synuclein and induces theformation of α-synuclein inclusion body.