title: Effect of oxidative stress on homer scaffolding proteins.
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.
authors: Ion Channel Research Unit and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
authors: Igor
authors: I
authors: Nepliouev
authors: Zhu-Shan
authors: ZS
authors: Zhang
authors: Jonathan A
authors: JA
authors: Stiber
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D002460:Cell Line; D004220:Disulfides; D000072233:Homer Scaffolding Proteins; D006801:Humans; D051379:Mice; D009135:Muscular Diseases / Q000209:etiology; D009154:Mutation; D009210:Myofibrils / Q000473:pathology; D019636:Neurodegenerative Diseases / Q000209:etiology; D018384:Oxidative Stress; D058105:Polymerization; D020033:Protein Isoforms; D012995:Solubility
chemical_list: D002352:Carrier Proteins; D004220:Disulfides; C000606614:HOMER1 protein, human; D000072233:Homer Scaffolding Proteins; D020033:Protein Isoforms