Molecular Interaction Between α-synuclein And Tubulin And Influence Of α-synuclein On The Polymerization Of Microtubule In Vitro

Alpha-synuclein(SNCP) is a kind of presynaptic proteins,distributing widely in brain tissues.Increasing evidences suggest thatα-synuclein is a common pathogenic molecule in several neurodegenerative diseases.The deposition of a-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion in these neurodegenerative disorders,which include Parkinson's disease,dementia with Lewy bodies,Lewy body variant of Alzheimer's disease,and multiple system atrophy, collectively referred to as synucleinopathies.Microtubules are cytoskeletal polymers consisting ofα/β-tubulin heterodimers and a variety of minor components known as microtubule-associated proteins(MAPs), which are important for the dynamic regulation and distribution ofmicrotubules in the cells.Microtubules also have a great contribution on the maintenance of the cell shape, the material transport as well as the mitosis and meiosis of the cell.It has been reported thatα-synuclein is a functional MAP,suggesting that further studies of the relationship between theα-synuclein and microtubules may provide a basis for understanding the structure and the function ofα-synuclein as well as the mechanisms of the neurodegeneration diseases.In this study,recombinant full-length human SNCP was expressed using prokaryotic expression system,and the native tubulin was extracted and purified from the bovine brain.Through pull-down and co-immunoprecipitation assays,a remarkable molecular interaction between the full-length SNCP and tubulin was identified.The co-location between SNCP and microtubule in cell was observed with confocal microscopy by immunofluorescence,which may provide evidences for the interaction between SNCP and microtubule in cell.To map the potential regions of SNCP for binding with tubulin,a serial of deleted mutations of SNCP were constructed and expressed.With pull-down and co-immunoprecipitation assays, clearly interactions of the peptides containing SNCP60-100,SNCP31-100 and SNCP1-100 were detected with tubulin,while the N terminus peptide SNCP1-60 failed.It demonstrated that the interaction regions within SNCP with tublin located at the peptide from aa 60 to 100.To test whether the interaction between SNCP and tubulin influences the assembling of microtubules from tubulin in vitro,microtubule assembly assay and sedimentation test were performed.It showed that the full-length SNCP influenced the microtubule dynamics as a tubulin-sequestering protein.Proteins SNCP60-100, SNCP31-100 and SNCP1-100 induced the same effect as the full-length SNCP in microtubule dynamics,while its N-terminal segments SNCP1-60 seemed not to affect microtubule polymerization in vitro.It suggested that the peptide SNCP60-100 may affect the assembling of microtubules from tubulin in vitro.