Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.
The human proteins FEZ1 and FEZ2 are orthologs of UNC‐76 from C. elegans, involved in growth and fasciculation of axons. Our aim is to exploit the molecular evolution of the FEZ protein family with emphasis on vertebrata branch. By the interactome profile of FEZ1 and FEZ2 from H. sapiens and UNC‐76 from C. elegans we observed a conserved pattern of protein‐protein interactions among FEZ1 and UNC‐76 that explain the ability of FEZ1 to rescue defects caused by unc‐76 mutations in nematodes. Furthermore, we characterized the interaction between FEZ1 and SCOCO (short coiled coil protein) by SAXS (Small Angle X‐ray Scattering). This interaction has been previously reported between their orthologs UNC‐76 and UNC‐69 that cooperate in axonal outgrowth. A hetero‐tetrameric state was observed, which consists of two GST‐SCOCO molecules attached to two FEZ1 molecules. By PAGE, SAXS, MS and NMR we defined that FEZ1 dimerizes by disulfide bond. In vivo this dimeric state could be important for kinesin mediated transport along microtubules. Thereby, FEZ1 may be classified as a dimeric and bivalent transport adaptor, essential to axon outgrowth and normal pre‐synaptic organization and transport of cargoes. The aggregation of new interaction partners found for the FEZ2 protein could be interpreted as the acquisition of new molecular functions and may have occurred in the early stages of chordate evolution. Supported by FAPESP, CNPq.
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