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Identification and Characterization of Gamma‐Synuclein as a PCBP1‐Interacting Protein in Various Cancer Cell Lines
Author(s) -
Ko Jane,
Hunkele Amanda,
Sultan Hamidah,
Ikalina Faith,
Liu Alexander,
NaharGohad Pranjal
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.802.3
Subject(s) - immunoprecipitation , microbiology and biotechnology , biology , ectopic expression , cdna library , complementary dna , expression vector , cell culture , chemistry , gene , biochemistry , recombinant dna , genetics
PolyC binding protein1 (PCBP1), a transcriptional regulator of mu‐opioid receptor (MOR) gene, has multiple functions, which can be regulated by various protein‐protein interactions. Using PCBP1 as bait, a human brain cDNA library was screened via two‐hybrid system. One candidate clone was identified as g‐synuclein with glutamic acid (E) at the 110 position rather than valine (V). It was called g‐synuclein110E in here to differentiate from the 110V form. The interaction between PCBP1 and g‐synuclein110E was confirmed by in vivo validation and GST‐pull down assay. Confocal analysis showed g‐synuclein110E mainly expressed in the cytosol of neuronal NMB cells. Ectopic expression of g‐synuclein110E or 110V did not alter MOR mRNA level or hMOR promoter activity, suggesting no involvement of g‐synuclein in modulating hMOR expression. Co‐immunoprecipitation using anti‐PCBP1 antibody and lysates of NMB cells overexpressing g‐synuclein110E or 110V revealed a stronger band intensity of co‐immunoprecipitated 110E form than 110V form, indicating that the change of amino acid at the 110 position affected the interaction with PCPB1. Synuclein110E form was found in H292 (lung), HT29 (colon) and T47D (breast) cells, and co‐immunoprecipitation demonstrated that this physical interaction with PCPB1 also occurred in these cells. To further examine if different endogenous g‐synuclein forms were also present in various cancer cells, RT‐PCR using RNAs from different cell lines and a specific primer set for g‐synuclein were used. The full length of g‐synuclein PCR products were cloned into a cloning vector and then several clones were subjected to DNA sequencing. Different sequences were detected in certain cell lines. Overall, this study reports g‐synuclein110E as a new PCBP1‐interacting protein and provides some insight into its complex role. Support or Funding Information Confocal microscope was acquired via the NSF grant. This research was supported by NIH research grant DA016673 and BioResearch Fund from Seton Hall University.