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Activity dependent dissociation of the Homer1 interactome.

ABSTRACT: Neurons encode information by rapidly modifying synaptic protein complexes, which changes the strength of specific synaptic connections. Homer1 is abundantly expressed at glutamatergic synapses, and is known to alter its binding to metabotropic glutamate receptor 5 (mGlu5) in response to synaptic activity. However, Homer participates in many additional known interactions whose activity-dependence is unclear. Here, we used co-immunoprecipitation and label-free quantitative mass spectrometry to characterize activity-dependent interactions in the cerebral cortex of wildtype and Homer1 knockout mice. We identified a small, high-confidence protein network consisting of mGlu5, Shank2 and 3, and Homer1-3, of which only mGlu5 and Shank3 were significantly reduced following neuronal depolarization. We identified several other proteins that reduced their co-association in an activity-dependent manner, likely mediated by Shank proteins. We conclude that Homer1 dissociates from mGlu5 and Shank3 following depolarization, but our data suggest that direct Homer1 interactions in the cortex may be more limited than expected.

SUBMITTER: Stillman M

PROVIDER: S-EPMC8881602 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Activity dependent dissociation of the Homer1 interactome.

Stillman Mason M Lautz Jonathan D JD Johnson Richard S RS MacCoss Michael J MJ Smith Stephen E P SEP

Scientific reports 20220225 1

Neurons encode information by rapidly modifying synaptic protein complexes, which changes the strength of specific synaptic connections. Homer1 is abundantly expressed at glutamatergic synapses, and is known to alter its binding to metabotropic glutamate receptor 5 (mGlu5) in response to synaptic activity. However, Homer participates in many additional known interactions whose activity-dependence is unclear. Here, we used co-immunoprecipitation and label-free quantitative mass spectrometry to ch ...[more]

PMID: 35217690

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Project description:BackgroundOxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process.MethodsA rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs).ResultsThe SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under β-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44.ConclusionsA novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Project description:Cellular structures are continually subjected to forces, which may serve as mechanical signals for cells through their effects on biomolecule interaction kinetics. Typically, molecular complexes interact via "slip bonds," so applied forces accelerate off rates by reducing transition energy barriers. However, biomolecules with multiple dissociation pathways may have considerably more complicated force dependencies. This is the case for DNA-binding proteins that undergo "facilitated dissociation," in which competitor biomolecules from solution enhance molecular dissociation in a concentration-dependent manner. Using simulations and theory, we develop a generic model that shows that proteins undergoing facilitated dissociation can form an alternative type of molecular bond, known as a "catch bond," for which applied forces suppress protein dissociation. This occurs because the binding by protein competitors responsible for the facilitated dissociation pathway can be inhibited by applied forces. Within the model, we explore how the force dependence of dissociation is regulated by intrinsic factors, including molecular sensitivity to force and binding geometry and the extrinsic factor of competitor protein concentration. We find that catch bonds generically emerge when the force dependence of the facilitated unbinding pathway is stronger than that of the spontaneous unbinding pathway. The sharpness of the transition between slip- and catch-bond kinetics depends on the degree to which the protein bends its DNA substrate. This force-dependent kinetics is broadly regulated by the concentration of competitor biomolecules in solution. Thus, the observed catch bond is mechanistically distinct from other known physiological catch bonds because it requires an extrinsic factor-competitor proteins-rather than a specific intrinsic molecular structure. We hypothesize that this mechanism for regulating force-dependent protein dissociation may be used by cells to modulate protein exchange, regulate transcription, and facilitate diffusive search processes.

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Activity dependent dissociation of the Homer1 interactome.

Publications

Activity dependent dissociation of the Homer1 interactome.

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