Abstract
Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.
Highlights
Homer proteins are a family of multifaceted scaffolding proteins that share a highly conserved Ena/VASP Homology 1 (EVH1) domain at their amino termini which allows binding to proline-rich motifs on Homer ligands which include group I metabotropic glutamate receptors, inositol triphosphate receptors (IP3R), the actin-binding protein Drebrin, and several members of the transient receptor potential (TRP) channel family [1,2,3]
We found by standard SDS-polyacrylamide gel electrophoresis (PAGE) of adult mouse skeletal muscle lysates exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent such as tris (2carboxyethyl) phosphine (TCEP) or beta-mercaptoethanol (BME) (Figure 1A)
WT and single cysteine mutants (C246G or C365G) exposed to air oxidation migrated as dimers with varying mobility based on the presence or absence of specific cysteine residues (Figure 2A)
Summary
Homer proteins are a family of multifaceted scaffolding proteins that share a highly conserved Ena/VASP Homology 1 (EVH1) domain at their amino termini which allows binding to proline-rich motifs on Homer ligands which include group I metabotropic glutamate receptors, inositol triphosphate receptors (IP3R), the actin-binding protein Drebrin, and several members of the transient receptor potential (TRP) channel family [1,2,3]. Homer 1a, which was identified as an immediate early gene (IEG), lacks a C-terminal coiled-coil domain [5]. Based on recently published crystallographic analysis of Homer 1 isoforms, Homer proteins form dimers via leucine zipper motifs at their C-terminal coiledcoil domains [7]. Homer tetramers form a polymeric network structure at the post synaptic density (PSD) through their interaction with other scaffolding proteins such as Shank, and this network is required for maintenance of dendritic spine structure and synaptic function [7]. A scaffolding protein complex involving Homer and Shank provides spatial organization to proteins involved in calcium signaling and links proteins involved in endocytosis and receptor recycling such as dynamin to the PSD [1,8]
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