Effect of Oxidative Stress on Homer Scaffolding Proteins

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

Oxidation results in disulfide cross-linking of Homer dimers.

<p>A) WT and mutant forms of Homer 1b were expressed in HEK 293 cells and exposed to air oxidation post lysis. WT and single cysteine mutants showed evidence of disulfide bond formation and migrated as dimers with varying mobility. With mutation of both cysteine residues (C246G, C365G) or addition of the reducing agent TCEP, Homer 1b migrated solely as a monomer. No bands were observed in lysates from untransfected control cells (CTL). B) HEK 293 cells were transfected with WT and mutant forms of Homer 1b, and cells were exposed to oxidative stress by addition of 200 µM menadione for 10 min. Oxidative stress resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). No bands were observed in lysates from untransfected control cells (CTL).</p

Oxidative modification of Homer results in loss of solubility.

<p>A) Western blot of lysates from C2C12 myotubes under control conditions (-) and conditions of oxidative stress (+200 µM menadione for 30 min.). Myotubes were lysed in 1% Triton and in the presence of 20 mM n-ethylmaleimide and run under non-reducing (−TCEP) and reducing conditions (+TCEP). B) Quantification of detectable Homer expression under control conditions (CTL) and after exposure to menadione (Mena) by non-reducing Western blot shown in (A). C) The addition of a reducing agent, either 10 mM reduced glutathione or 10 mM DTT, to the media prior to menadione exposure successfully blocked the loss in detectable Homer observed after prolonged (1 hour) exposure to menadione (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026128#pone-0026128-g003" target="_blank">Figure 3C</a>). D) Adult myofibers were exposed to control conditions or oxidative stress by addition of 200 µM H₂O₂. Western blot of Homer protein expression showing a decrease in detectable Homer in the Triton soluble fraction in response to oxidative stress and an increase in Homer detected in Triton insoluble fraction. E) Lysates from HEK 293 cells transfected with WT Homer 1b or the double mutant (C246G, C365G) and exposed to intracellular oxidative stress (200 µM menadione for 30 min.) were separated into Triton soluble and insoluble fractions. WT Homer 1b was detected in the Triton insoluble fraction only in response to oxidative stress, but no evidence of the double mutant (C246G, C365G) was detected in the insoluble fraction in response to oxidative stress. F) C2C12 myotubes were exposed to 200 µM menadione for 0, 30, and 60 minutes respectively. Cells were lysed in buffer containing 1% Triton, 8 M guanidine HCL, and 50 mM TCEP. Analysis of whole lysates showed a decrease in detectable Homer over time.</p

Effect of oxidative modification on the <i>in vitro</i> interaction of Homer and Drebrin.

<p>A) Recombinant Homer 1a was expressed as a GST fusion protein in E. coli and purified. We saw a significant <i>in vitro</i> interaction between recombinant Homer 1a and recombinant Drebrin using an ELISA format. GST protein alone served as a negative control and showed an insignificant interaction with Drebrin. W27A mutation of the EVH1 domain of Homer 1a significantly inhibited the interaction of Homer 1a-GST and Drebrin. B) Mutation of the first (F543A) or second (F621A) or both (F543A, F621A) Homer binding motifs (PPxxF) at the carboxyl terminus of Drebrin significantly inhibited the interaction of recombinant Homer 1a and Drebrin <i>in vitro</i> as measured by ELISA. C) <i>In vitro</i> binding of recombinant Homer 1b to Drebrin. Urea (2 M) had no effect on binding, while reduction of disulfide bonds with DTT (50 mM) or S-methylthiolation of cysteine resides with MMTS (10 mM) significantly blocked binding of recombinant Homer 1b to Drebrin. D) Mutation of both cysteine residues of Homer 1b resulted in a significant decrease in binding to Drebrin compared to WT Homer 1b.</p

Immunoblotting of skeletal muscle lysates exposed to air oxidation.

<p>A) Western blot of adult mouse skeletal muscle lysates showing that Homer migrates as both a dimer and monomer in the absence of reducing agent (lane 1) and solely as a monomer in the presence of reducing agent (BME, lane 2). Neither the 90 kDa or 45 kDa band was observed in skeletal muscle lysates from Homer 1 knockout mice. B) Homer 1b protein sequence showing the location of the two cysteine residues (residues 246 and 365).</p

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple TRPC channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: WT and congenic Dbn(−/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4 ± 0.3-fold greater in Dbn(−/+) than in WT mice. Levels of G-actin were equivalent in Dbn(−/+) and WT SMCs, but there was a 2.4 ± 0.5-fold decrease in F-actin in Dbn(−/+) SMCs compared with WT. F-actin was restored to WT levels in Dbn(−/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(−/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(−/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation though its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds