ABSTRACT
BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.
Subject(s)
Apoptosis , Voltage-Dependent Anion Channel 2 , bcl-2 Homologous Antagonist-Killer Protein , Voltage-Dependent Anion Channel 2/metabolism , Voltage-Dependent Anion Channel 2/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , Humans , Protein Binding , Mitochondria/metabolism , Animals , HEK293 CellsABSTRACT
The common neurodegenerative disorder Alzheimer disease (AD) is characterized by memory dysfunction and cognitive decline in the elderly. Neuropathological features include aggregated ß-amyloid (Aß) accumulation, neuroinflammation, and oxidative stress in the brain. Daphnetin (DAPH), a natural coumarin derivative, has the potential for inhibiting inflammatory and oxidative responses. We explored neuroprotective roles of DAPH treatment in the APP/PS1 transgenic mouse AD model. DAPH ameliorated spatial learning disabilities in Morris water maze tests and reduced Aß deposition, assessed by immunohistochemistry. It also reduced the Aß content in supernatants of neurons from fetal APP/PS1 mice, assessed by cell-based soluble ELISA. Molecular docking and fluorescence resonance energy transfer-based assay results suggested that DAPH could directly inhibit BACE1 activity. Furthermore, in vitro experiments utilizing isolated rat neurons assessing RNA expression profiling, immunofluorescence, TUNEL assay, and Western-blot analysis, suggested the potential of DAPH for regulating BDNF and GM-CSF expression and mitigating Aß1-42-induced cortical injury, synaptic loss, and apoptosis. HO-1 and Nrf2 mRNA and protein expression were also increased in a dose-dependent manner. These results underscore the potential of DAPH as a neuroprotective agent in reversing memory deficits associated with AD and bolster its candidacy as a multitarget natural small-molecule drug for AD patients.
Subject(s)
Alzheimer Disease , Amyloid Precursor Protein Secretases , Aspartic Acid Endopeptidases , Disease Models, Animal , Heme Oxygenase-1 , Mice, Transgenic , NF-E2-Related Factor 2 , Neurons , Neuroprotective Agents , Umbelliferones , Animals , Alzheimer Disease/pathology , Alzheimer Disease/metabolism , Alzheimer Disease/drug therapy , NF-E2-Related Factor 2/metabolism , Umbelliferones/pharmacology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Amyloid Precursor Protein Secretases/metabolism , Rats , Aspartic Acid Endopeptidases/metabolism , Mice , Neuroprotective Agents/pharmacology , Heme Oxygenase-1/metabolism , Signal Transduction/drug effects , Amyloid beta-Peptides/metabolism , Rats, Sprague-Dawley , Male , Membrane ProteinsABSTRACT
OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.
Subject(s)
Calcium Channels , Cryoelectron Microscopy , Ion Channel Gating , Mechanotransduction, Cellular , Humans , Anoctamins/chemistry , Anoctamins/metabolism , Calcium Channels/chemistry , Calcium Channels/metabolism , Calcium Channels/ultrastructure , Lipids/chemistry , Liposomes/metabolism , Liposomes/chemistry , Models, Molecular , Nanostructures/chemistryABSTRACT
Terpenoids constitute the largest class of plant primary and secondary metabolites with a broad range of biological and ecological functions. They are synthesized from isopentenyl diphosphate and dimethylallyl diphosphate, which in plastids are condensed by geranylgeranyl diphosphate synthases (GGPPSs) to produce GGPP (C20) for diterpene biosynthesis and by geranyl diphosphate synthases (GPPSs) to form GPP (C10) for monoterpene production. Depending on the plant species, unlike homomeric GGPPSs, GPPSs exist as homo- and heteromers, the latter of which contain catalytically inactive GGPPS-homologous small subunits (SSUs) that can interact with GGPPSs. By combining phylogenetic analysis with functional characterization of GGPPS homologs from a wide range of photosynthetic organisms, we investigated how different GPPS architectures have evolved within the GGPPS protein family. Our results reveal that GGPPS gene family expansion and functional divergence began early in nonvascular plants, and that independent parallel evolutionary processes gave rise to homomeric and heteromeric GPPSs. By site-directed mutagenesis and molecular dynamics simulations, we also discovered that Leu-Val/Val-Ala pairs of amino acid residues were pivotal in the functional divergence of homomeric GPPSs and GGPPSs. Overall, our study elucidated an evolutionary path for the formation of GPPSs with different architectures from GGPPSs and uncovered the molecular mechanisms involved in this differentiation.
Subject(s)
Dimethylallyltranstransferase , Diterpenes , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , Phylogeny , Dimethylallyltranstransferase/genetics , Dimethylallyltranstransferase/metabolism , Diterpenes/metabolismABSTRACT
Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.
Subject(s)
Ion Channel Gating , Membrane Lipids/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Potassium/metabolism , Anions/chemistry , Anions/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Ion Transport , Liposomes/chemistry , Liposomes/metabolism , Membrane Lipids/chemistry , Molecular Dynamics Simulation , Mutation , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylserines/chemistry , Phosphatidylserines/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/geneticsABSTRACT
The primary means by which ion permeation through potassium channels is controlled, and the key to selective intervention in a range of pathophysiological conditions, is the process by which channels switch between non-conducting and conducting states. Conventionally, this has been explained by a steric mechanism in which the pore alternates between two conformations: a 'closed' state in which the conduction pathway is occluded and an 'open' state in which the pathway is sufficiently wide to accommodate fully hydrated ions. Recently, however, 'non-canonical' mechanisms have been proposed for some classes of K+ channels. The purpose of this review is to illuminate structural and dynamic relationships underpinning permeation control in K+ channels, indicating where additional data might resolve some of the remaining issues.
Subject(s)
Potassium Channels , Potassium , Potassium/metabolism , Potassium Channels/metabolism , Protein ConformationABSTRACT
The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.
Subject(s)
G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Potassium/metabolism , Cytosol/chemistry , Cytosol/metabolism , Electric Conductivity , Electric Impedance , G Protein-Coupled Inwardly-Rectifying Potassium Channels/chemistry , G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics , Humans , Ion Transport , Ions/chemistry , Ions/metabolism , Molecular Dynamics Simulation , Potassium/chemistry , Protein Conformation , Water/metabolismABSTRACT
The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target.