RESUMEN
Vacuolar-type adenosine triphosphatases (V-ATPases) not only function as rotary proton pumps in cellular organelles but also serve as signaling hubs. To identify the endogenous binding partners of V-ATPase, we collected a large dataset of human V-ATPases and did extensive classification and focused refinement of human V-ATPases. Unexpectedly, about 17% of particles in state 2 of human V-ATPases display additional density with an overall resolution of 3.3 Å. Structural analysis combined with artificial intelligence modeling enables us to identify this additional density as mEAK-7, a protein involved in mechanistic target of rapamycin (mTOR) signaling in mammals. Our structure shows that mEAK-7 interacts with subunits A, B, D, and E of V-ATPases in state 2. Thus, we propose that mEAK-7 may regulate V-ATPase function through binding to V-ATPases in state 2 as well as mediate mTOR signaling.
Asunto(s)
Microscopía por Crioelectrón , Minería de Datos , ATPasas de Translocación de Protón Vacuolares , Animales , Inteligencia Artificial , Microscopía por Crioelectrón/métodos , Minería de Datos/métodos , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Unión Proteica/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
OBJECTIVE: Mutations in KCNC1 can cause severe neurological dysfunction, including intellectual disability, epilepsy, and ataxia. The Arg320His variant, which occurs in the voltage-sensing domain of the channel, causes a highly penetrant and specific form of progressive myoclonus epilepsy with severe ataxia, designated myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK). KCNC1 encodes the voltage-gated potassium channel KV 3.1, a channel that is important for enabling high-frequency firing in interneurons, raising the possibility that MEAK is associated with reduced interneuronal function. METHODS: To determine how this variant triggers MEAK, we expressed KV 3.1bR320H in cortical interneurons in vitro and investigated the effects on neuronal function and morphology. We also performed electrophysiological recordings of oocytes expressing KV 3.1b to determine whether the mutation introduces gating pore currents. RESULTS: Expression of the KV 3.1bR320H variant profoundly reduced excitability of mature cortical interneurons, and cells expressing these channels were unable to support high-frequency firing. The mutant channel also had an unexpected effect on morphology, severely impairing neurite development and interneuron viability, an effect that could not be rescued by blocking KV 3 channels. Oocyte recordings confirmed that in the adult KV 3.1b isoform, R320H confers a dominant negative loss-of-function effect by slowing channel activation, but does not introduce potentially toxic gating pore currents. SIGNIFICANCE: Overall, our data suggest that, in addition to the regulation of high-frequency firing, KV 3.1 channels play a hitherto unrecognized role in neuronal development. MEAK may be described as a developmental dendritopathy.
Asunto(s)
Dendritas/patología , Epilepsias Mioclónicas Progresivas/fisiopatología , Neurogénesis/genética , Canales de Potasio Shaw/genética , Animales , Humanos , Interneuronas/patología , Ratones , Ratones Endogámicos C57BL , Mutación , Epilepsias Mioclónicas Progresivas/genéticaRESUMEN
mEAK-7 (mammalian EAK-7 or MTOR-associated protein, eak-7 homolog), is an evolutionarily conserved lysosomal membrane protein that is highly expressed in several cancer cells. Multiple recent studies have identified mEAK-7 as a positive activator of mTOR (mammalian/mechanistic target of rapamycin) signaling via an alternative mTOR complex, implying that mEAK-7 plays an important role in the promotion of cancer proliferation and migration. In addition, structural analyses investigating interactions between mEAK-7 and V-ATPase, a protein complex responsible for regulating pH homeostasis in cellular compartments, have suggested that mEAK-7 may contribute to V-ATPase-mediated mTORC1 activation. The C-terminal α-helix of mEAK-7 binds to the D and B subunits of the V-ATPase, creating a pincer-like grip around its B subunit. This binding undergoes partial disruption during ATP hydrolysis, potentially enabling other proteins such as mTOR to bind to the α-helix of mEAK-7. mEAK-7 also promotes chemoresistance and radiation resistance by sustaining DNA damage-mediated mTOR signaling through interactions with DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Taken together, these findings indicate that mEAK-7 may be a promising therapeutic target against tumors. However, the precise molecular mechanisms and signal transduction pathways of mEAK-7 in cancer remain largely unknown, motivating the need for further investigation. Here, we summarize the current known roles of mEAK-7 in normal physiology and cancer development by reviewing the latest studies and discuss potential future developments of mEAK-7 in targeted cancer therapy.
RESUMEN
Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.
Asunto(s)
Proteínas Activadoras de GTPasa , ATPasas de Translocación de Protón Vacuolares , Humanos , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/química , Coactivadores de Receptor Nuclear/metabolismo , Coactivadores de Receptor Nuclear/química , Unión Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas MitocondrialesRESUMEN
Progressive myoclonic epilepsy (PME) is characterized by prominent myoclonus and generalized or focal seizures. A recently described novel KCNC1 mutation is associated with a specific phenotype of progressive myoclonic epilepsy, which has been defined as myoclonic epilepsy and ataxia due to potassium channel mutation (MEAK). Our case illustrates a typical presentation of this disease and the potential for misdiagnosis as idiopathic generalized epilepsy during the early phase of the disease. Unique findings that may suggest an alternative diagnosis are a progressive myoclonus, prominent ataxia/dysmetria on examination, and abnormally high amplitude in the sensory evoked potential recording. We also report a brief review of the existing literature on MEAK. Early and accurate diagnosis with genetic testing may significantly help in counseling patients and families.
Asunto(s)
Epilepsias Mioclónicas Progresivas/diagnóstico , Epilepsias Mioclónicas Progresivas/fisiopatología , Canales de Potasio Shaw/genética , Adolescente , Ataxia/diagnóstico , Ataxia/genética , Ataxia/fisiopatología , Electroencefalografía , Epilepsia Generalizada/diagnóstico , Pruebas Genéticas , Humanos , Imagen por Resonancia Magnética , Masculino , Epilepsias Mioclónicas Progresivas/genéticaRESUMEN
Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modification. Recently, mEAK-7 (mTOR associated protein, eak-7 homolog) was identified as a positive activator of mTOR signaling via an alternative mTOR complex. However, the upstream regulation of mEAK-7 in human cells is not known. Because microRNAs are capable of modulating protein translation of RNA in eukaryotes, we conducted a bioinformatic search for relevant mEAK-7 targeting microRNAs using the Exiqon miRSearch V3.0 algorithm. Based on the score obtained through miRSearch V3.0, the top predicted miRNA (miR-1911-3p) was studied. miR-1911-3p mimics decreased protein levels of both mEAK-7 and mTORC1 downstream effectors p-S6 and p-4E-BP1 in non-small cell lung carcinoma (NSCLC) cell lines H1975 and H1299. miR-1911-3p levels and MEAK7 mRNA/mEAK-7/mTOR signaling levels were negatively correlated between normal lung and NSCLC cells. miR-1911-3p directly interacted with MEAK7 mRNA at the 3'-UTR to negatively regulate mEAK-7 and significantly decreased mTOR localization to the lysosome. Furthermore, miR-1911-3p significantly decreased cell proliferation and migration in both H1975 and H1299 cells. Thus, miR-1911-3p functions as a suppressor of mTOR signaling through the regulation of MEAK7 mRNA translation in human cancer cells.
RESUMEN
Progressive myoclonus epilepsies (PMEs) constitute a cluster of inherent, genetically diverse, rare seizure disorders characterized by ataxia, tonic-clonic seizures, and action myoclonus. Recently, a mutation in the KCNC1 gene (Arg320His) was described in a group of PME patients. The KCNC1 gene encodes the Kv3.1 potassium ion channel responsible for the rapid repolarization of the membrane potential following action potential firing in fast spiking GABAergic interneurons (FSI), thereby enabling high firing frequency. In the present study, we demonstrate that the Arg320His mutation cause a reduction in the Kv3.1 current amplitude and acts in a dominantly negative fashion. The mutation profoundly affects channel activation and deactivation kinetics, and we further find that it impairs recruitment of the Kv3.1 channel to the plasma membrane. The Kv3 activating compound, RE01, partly rescues the electrophysiological deficit, suggesting that pharmacological activation of Kv3.1 activity might be a feasible approach for treatment of this cohort of PME patients.
Asunto(s)
Hidantoínas/farmacología , Epilepsias Mioclónicas Progresivas/tratamiento farmacológico , Piridinas/farmacología , Canales de Potasio Shaw/metabolismo , Potenciales de Acción/efectos de los fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Hidantoínas/uso terapéutico , Mutagénesis Sitio-Dirigida , Epilepsias Mioclónicas Progresivas/genética , Técnicas de Placa-Clamp , Piridinas/uso terapéutico , Canales de Potasio Shaw/genética , TransfecciónRESUMEN
Progressive myoclonus epilepsy (PME) is a distinct group of seizure disorders characterized by gradual neurological decline with ataxia, myoclonus and recurring seizures. There are several forms of PME, among which the most recently described is MEAK - myoclonus epilepsy and ataxia due to potassium channel mutation. This particular subtype is caused by a recurrent de novo heterozygous mutation (c.959G>A, p.Arg320His) in the KCNC1 gene, which maps to chromosome 11 and encodes for the Kv3.1 protein (a subunit of the Kv3 subfamily of voltage-gated potassium channels). Loss of Kv3 function disrupts the firing properties of fast-spiking neurons, affects neurotransmitter release and induces cell death. Specifically regarding Kv3.1 malfunctioning, the most affected neurons include inhibitory GABAergic interneurons and cerebellar neurons. Impairment of the former cells is believed to contribute to myoclonus and seizures, whereas dysfunction of the latter to ataxia and tremor. Phenotypically, MEAK patients generally have a normal early development. At the age of 6 to 14 years, they present with myoclonus, which tends to progressively worsen with time. Tonic-clonic seizures may or may not be present, and some patients develop mild cognitive impairment following seizure onset. Typical electroencephalographic features comprise generalized epileptiform discharges and, in some cases, photosensitivity. Brain imaging is either normal or shows cerebellar atrophy. The identification of MEAK has both expanded the phenotypic and genotypic spectra of PME and established an emerging role for de novo mutations in PME.