Mamais, A.; (2010) Investigation of an N5-glutamine methyltranferase, a novel partner of α2-chimaerin. Doctoral thesis , UCL (University College London).

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Abstract

Differentiating neurones respond to extracellular signalling cues that affect the actin cytoskeleton and influence the outgrowth and retraction of cellular processes. Rac1 belongs to the family of Rho GTPases that play a key role in actin organisation during neuronal development. Rac1 promotes lamellipodia formation and neurite outgrowth, and is also involved in axonal retraction pathways. α2-Chimaerin down-regulates Rac1 and is involved in neuronal plasticity and axonal guidance through a wide spectrum of interacting partners. The N5-glutamine methyltransferase HemK1 was previously identified in our lab as a novel interacting partner of α2-chimaerin, in a yeast two-hybrid screen. The aim of this study was to characterise HemK1 and investigate its role in neurite outgrowth. N5-glutamine methyltransferases are universally conserved in nature and little studied in vertebrates. HemK1 and related protein HemK2 are implicated in the control of translation termination by methylating the polypeptide chain release factors, a modification that mediates efficient translation termination in their bacterial and yeast homologues. Analyses of their transcript levels in rat embryonic brains by quantitative real-time PCR indicated that both HemK1 and HemK2 are expressed at comparable levels to α2-chimaerin in the brain and also in hippocampal neurones. HemK1 monoclonal antibodies detected an endogenous protein in brain mitochondrial fractions, but not in cytosol. When over-expressed, HemK1 co-localised with its proposed mitochondrial substrate mtRF1a in cells and also exhibited partial colocalisation with Dcp1b, a component of the mRNA decay machinery. Both HemK1 and HemK2 associated with α2-chimaerin, as well as with their proposed substrates, mtRF1a and eRF1. α2-Chimaerin influences neuronal morphology and dendritic pruning. ShRNA knock-down of HemK1 or HemK2 in primary rat hippocampal neurones in culture promoted increased branching and complexity of neurites as assessed by confocal microscopy and Sholl analysis. These results suggest a novel link between translational control mechanisms and Rac signalling pathways in developing neurones.

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