Identification of gamma-synuclein as a new PCBP1-interacting protein

Abstract

Objectives: PolyC binding protein 1 (PCBP1) is a transcriptional regulator of human mu-opioid receptor (hMOR) gene in the CNS and is also related to cancer/diseases. It possesses multi-roles that can be mediated by protein-protein interactions. To understand the mechanism controlling PCBP1 functions, PCBP1-interacting protein was investigated.

Methods: Using PCBP1 as the bait, a human brain cDNA library was screened via two-hybrid system. DNA sequence of candidate protein was confirmed using NCBI/SNP databases. Candidate protein in various cell lines was examined by RT-PCR. Glutathione-S-transferase (GST) pull-down and co-immunoprecipitation were used to validate the physical interaction. Its effects on hMOR gene regulation were examined.

Results: One clone was identified as gamma-synuclein110E, an SNP of gamma-synuclein110V. The interaction between PCBP1 and gamma-synuclein110E was confirmed by further validation and GST pull-down assay. Confocal analysis showed gamma-synuclein110E mainly expressing in the cytosol of human neuronal NMB cells. This interaction was confirmed by co-immunoprecipitation with NMB lysates, containing both proteins endogenously. Ectopic expression of gamma-synuclein110E or 110V did not alter hMOR mRNA level or promoter activity, suggesting no involvement of gamma-synuclein in modulating hMOR expression. Co-immunoprecipitation using gamma-synuclein110E or 110V overexpressed NMB cells with anti-PCBP1 antibody revealed a stronger intensity of co-immunoprecipitated gamma-synuclein band using gamma-synuclein110E-overexpressed cells as compared to that using gamma-synuclein110V-overexpressed cells. Synuclein110E was also identified in H292 (lung), HT29 (colon) and T47D (breast) cells, and this physical interaction was confirmed.

Conclusion: We report a newly identified PCBP1-interacting protein, gamma-synuclein110E, and provide some insight into its complex role as well as discuss potential roles of this interaction.

Keywords: Gamma-synuclein; H292; HT29 and T47D cells; Human NMB; Human brain cDNA library; PCBP1; Physical interaction; SNP; Two-hybrid screening.