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


CONCLUSIONS
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.
a Department of Biological Sciences ; Seton Hall University , USA.
Amanda
A
Hunkele
a Department of Biological Sciences ; Seton Hall University , USA.
Hamidah
H
Sultan
a Department of Biological Sciences ; Seton Hall University , USA.
Faith A
FA
Ikalina
a Department of Biological Sciences ; Seton Hall University , USA.
Alexander H
AH
Liu
a Department of Biological Sciences ; Seton Hall University , USA.
Pranjal
P
Nahar-Gohad
a Department of Biological Sciences ; Seton Hall University , USA.
Jane L
JL
Ko
D001921:Brain / Q000378:metabolism*; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins; D015723:Gene Library; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D009447:Neuroblastoma / Q000473:pathology; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D060066:Protein Interaction Maps; D016601:RNA-Binding Proteins; D014162:Transfection; D020798:Two-Hybrid System Techniques; D051845:gamma-Synuclein / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C094604:PCBP1 protein, human; D016601:RNA-Binding Proteins; D051845:gamma-Synuclein
Gamma-synuclein; H292; HT29 and T47D cells; Human NMB; Human brain cDNA library; PCBP1; Physical interaction; SNP; Two-hybrid screening