The effect of dopamine on the interaction between the FOXP2 FHD and DNA
Date
2020
Authors
Symon, Joni
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Abstract
Transcription factors play a crucial role in the regulation of both basal and differential gene expression, interacting with specific target sequences, thereby activating or repressing transcription. The FOX family of transcription factors, characterised by the presence of a modified helix-turn-helix DNA binding domain, the forkhead domain (FHD), are key in the regulation of pathways associated with embryogenesis and development. One such member, FOXP2 has been directly associated with language acquisition. Mutations, specifically with in the FHD, have been directly linked to verbal dyspraxia and
cancer, as well as hypothesized links between abnormal FOXP2 function and autism and schizophrenia . It is possible that the function of FOXP2 could be regulated by the presence of small molecules, specifically dopamine. Various studies have suggested links between dopaminergic pathways and FOXP2 activity. For example, it has been proposed that FOXP2 down regulates cellular dopamine levels, as well as affects the morphologies of target neurons of dopaminergic pathways. The potential for the neurotransmitter to directly interact with FOXP2, specifically the FHD, there by modulating the activity of the protein, specifically with regard to DNA biding, had not yet been investigated . FOX P2 FHD was over expressed, purified and structurally and functionally characterised, before investigating whether the presence of dopamine at physiological concentrations (35 M ), as well as in (100 M ), affected the secondary or tertiary structure. The effect of the presence of the neurotransmitter on DNA binding was then investigated, and the KD was determined. Molecular docking was used to investigate the potential for an interaction between the FOXP2 FHD and dopamine, as well as the FHD and various reactive products of dopamine autoxidation (namely 5, 6-dihydroxyindole, 5, 6-indolequinone a n d dopaminechrome ) in silico. The investigation revealed a potential non-canonical binding pocket located towards the C-terminus of the FOXP2 FHD, however, it appeared that a product of dopamine autoxidation, such as 5, 6-dihydroxyindole, was more likely to bind the pocket than dopamine itself. Spectroscopic investigation revealed that the presence of dopamine did not alter the fold or conformation of the protein, while fluorescence anisotropy suggested that the neurotransmitter did not significantly affect the DNA binding affinity, however a slight reduction in binding affinity was observed with increasing concentrations of dopamine (a KD of 0, 784M was observed in the absence of dopamine, while the KD obtained in the presence of 35 M dopamine was found to be 1, 074 M, and in the presence of 100 M dopamine, the KD obtained was 1 , 40 M). Supported by in silico observations, it was hypothesized that it is unlikely that dopamine itself directly regulates the activity of FOXP2 via interactions with the FHD, however it is possible that a product of spontaneous dopamine autoxidation may play a role in regulating FOXP2 activity physiologically
Description
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science, 2020