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. 2014 May;34(4):603-9.
doi: 10.1007/s10571-014-0046-9.

α-Synuclein is prone to interaction with the GC-box-like sequence in vitro

Kai-Li Ma, Lian-Kun Song, Yu-He Yuan, Ying Zhang, Jin-Ling Yang, Ping Zhu, Nai-Hong Chen

α-Synuclein is prone to interaction with the GC-box-like sequence in vitro

Kai-Li Ma et al. Cell Mol Neurobiol. 2014 May.

Abstract

α-Synuclein (α-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinson's disease, a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons and the formation of Lewy bodies. Evidence suggests that α-syn could be imported into the nucleus and subsequently disrupt normal neuronal function. The existence of α-syn in the nucleus provides the possibility of interaction with DNA leading to gene transcript regulation. Thus, CD spectra were used to determine the specific DNA sequence with which α-syn is most likely to interact. Our results indicated that α-syn was prone to preferentially interact with the GC-box-like sequence in vitro at a ratio of 2:1 or less (α-syn: the GC-box-like sequence).

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig. 1
Fig. 1
The expression and purification of α-syn-GFP protein. a The separated α-syn-GFP protein using anion exchange chromatography was subjected to SDS-PAGE gel electrophoresis and visualized via staining with Bromophenol Blue. Lane 1 markers (in kDa from the highest to lowest): 72, 55, 43, 34, 26, and 17. Lane 2 α-syn-GFP protein. b Western blot analysis for α-syn-GFP was performed. A mouse monoclonal antibody specific for α-syn (Santa Cruz) was used at a dilution of 1:500
Fig. 2
Fig. 2
Circular dichroism spectra show α-syn binding. a The binding assay between the purified α-syn-GFP protein and the DNA sequences rich in bases AG or TC. b The binding assay between α-syn and the CCAT-box-like sequence or GC-box-like sequence. Four scans were taken and averaged from 410 to 230 nm, and the data were collected every 0.5 nm. Representative spectra are shown, corresponding to protein: DNA ratios of 1:1. These experiments were carried out in 50 mM Tris-HCI, 5 mM MgCl2, 100 mM NaCI, pH 8.0 at 20 °C in a 4-mm path length cell
Fig. 3
Fig. 3
Circular dichroism spectra showing α-syn binding. a Result from the binding assay between the purified α-syn-GFP protein and the GC-box-like DNA sequence. b The binding assay results between the commercial α-syn protein and the GC-box-like DNA sequence. c The binding assay results between the commercial GFP protein and the GC-box-like DNA sequence. Four scans were taken and averaged from 410 to 230 nm, and the data were collected every 0.5 nm. Representative spectra are shown, corresponding to protein: DNA ratios of a 0.5:1, 1:1, 2:1, 3:1, and 4:1; b 1:1, 2:1, 3:1, and 4:1; and c 1:1, 2:1, 3:1, and 4:1. These experiments were carried out in 50 mM Tris-HCI, 5 mM MgCl2, 100 mM NaCI, pH 8.0 at 20 °C in a 4-mm path length cell
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