RESUMEN
Parthanatos-associated apoptosis-inducing factor (AIF) nuclease (PAAN), also known as macrophage migration inhibitor factor (MIF), is a member of the PD-D/E(X)K nucleases that acts as a final executioner in parthanatos. PAAN's role in Parkinson's disease (PD) and whether it is amenable to chemical inhibition is not known. Here, we show that neurodegeneration induced by pathologic α-synuclein (α-syn) occurs via PAAN/MIF nuclease activity. Genetic depletion of PAAN/MIF and a mutant lacking nuclease activity prevent the loss of dopaminergic neurons and behavioral deficits in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Compound screening led to the identification of PAANIB-1, a brain-penetrant PAAN/MIF nuclease inhibitor that prevents neurodegeneration induced by α-syn PFF, AAV-α-syn overexpression, or MPTP intoxication in vivo. Our findings could have broad relevance in human pathologies where parthanatos plays a role in the development of cell death inhibitors targeting the druggable PAAN/MIF nuclease.
Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Enfermedad de Parkinson , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Endonucleasas/metabolismo , Ratones , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.
Asunto(s)
Microcefalia , Trastornos del Movimiento , Trastornos del Neurodesarrollo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células HEK293 , Serina-Treonina Quinasas TORRESUMEN
Clinical reports suggest that the coronavirus disease-19 (COVID-19) pandemic caused by severe acute respiratory syndrome (SARS)-coronavirus-2 (CoV-2) has not only taken millions of lives, but has also created a major crisis of neurologic complications that persist even after recovery from the disease. Autopsies of patients confirm the presence of the coronaviruses in the CNS, especially in the brain. The invasion and transmission of SARS-CoV-2 in the CNS is not clearly defined, but, because the endocytic pathway has become an important target for the development of therapeutic strategies for COVID-19, it is necessary to understand endocytic processes in the CNS. In addition, mitochondria and mechanistic target of rapamycin (mTOR) signaling pathways play a critical role in the antiviral immune response, and may also be critical for endocytic activity. Furthermore, dysfunctions of mitochondria and mTOR signaling pathways have been associated with some high-risk conditions such as diabetes and immunodeficiency for developing severe complications observed in COVID-19 patients. However, the role of these pathways in SARS-CoV-2 infection and spread are largely unknown. In this review, we discuss the potential mechanisms of SARS-CoV-2 entry into the CNS and how mitochondria and mTOR pathways might regulate endocytic vesicle-mitochondria interactions and dynamics during SARS-CoV-2 infection. The mechanisms that plausibly account for severe neurologic complications with COVID-19 and potential treatments with Food and Drug Administration-approved drugs targeting mitochondria and the mTOR pathways are also addressed.
Asunto(s)
COVID-19/complicaciones , Enfermedades del Sistema Nervioso/virología , Neuronas/virología , Animales , COVID-19/metabolismo , COVID-19/patología , COVID-19/virología , Humanos , Mitocondrias/metabolismo , Mitocondrias/virología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , Neuronas/metabolismo , SARS-CoV-2/patogenicidad , Serina-Treonina Quinasas TOR/metabolismo , Síndrome Post Agudo de COVID-19 , Tratamiento Farmacológico de COVID-19RESUMEN
Mutations in the IQSEC2 gene are associated with drug-resistant, multifocal infantile and childhood epilepsy; autism; and severe intellectual disability (ID). We used induced pluripotent stem cell (iPSC) technology to obtain hippocampal neurons to investigate the neuropathology of IQSEC2-mediated disease. The neurons were characterized at three-time points during differentiation to assess developmental progression. We showed that immature IQSEC2 mutant dentate gyrus (DG) granule neurons were extremely hyperexcitable, exhibiting increased sodium and potassium currents compared to those of CRISPR-Cas9-corrected isogenic controls, and displayed dysregulation of genes involved in differentiation and development. Immature IQSEC2 mutant cultured neurons exhibited a marked reduction in the number of inhibitory neurons, which contributed further to hyperexcitability. As the mutant neurons aged, they became hypoexcitable, exhibiting reduced sodium and potassium currents and a reduction in the rate of synaptic and network activity, and showed dysregulation of genes involved in synaptic transmission and neuronal differentiation. Mature IQSEC2 mutant neurons were less viable than wild-type mature neurons and had reduced expression of surface AMPA receptors. Our studies provide mechanistic insights into severe infantile epilepsy and neurodevelopmental delay associated with this mutation and present a human model for studying IQSEC2 mutations in vitro.
Asunto(s)
Trastorno Autístico , Epilepsia , Discapacidad Intelectual , Anciano , Trastorno Autístico/genética , Niño , Epilepsia/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Discapacidad Intelectual/genética , Mutación/genética , Neuronas/metabolismo , Transmisión Sináptica/genéticaRESUMEN
Members of the AAA+ superfamily of ATPases are involved in the unfolding of proteins and disassembly of protein complexes and aggregates. ATAD1 encoding the ATPase family, AAA+ domain containing 1-protein Thorase plays an important role in the function and integrity of mitochondria and peroxisomes. Postsynaptically, Thorase controls the internalization of excitatory, glutamatergic AMPA receptors by disassembling complexes between the AMPA receptor-binding protein, GRIP1, and the AMPA receptor subunit GluA2. Using whole-exome sequencing, we identified a homozygous frameshift mutation in the last exon of ATAD1 [c.1070_1071delAT; p.(His357Argfs*15)] in three siblings who presented with a severe, lethal encephalopathy associated with stiffness and arthrogryposis. Biochemical and cellular analyses show that the C-terminal end of Thorase mutant gained a novel function that strongly impacts its oligomeric state, reduces stability or expression of a set of Golgi, peroxisomal and mitochondrial proteins and affects disassembly of GluA2 and Thorase oligomer complexes. Atad1-/- neurons expressing Thorase mutantHis357Argfs*15 display reduced amount of GluA2 at the cell surface suggesting that the Thorase mutant may inhibit the recycling back and/or reinsertion of AMPA receptors to the plasma membrane. Taken together, our molecular and functional analyses identify an activating ATAD1 mutation as a new cause of severe encephalopathy and congenital stiffness.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/genética , Encefalopatías/genética , Regulación de la Expresión Génica/genética , Mutación/genética , Neuronas/patología , Receptores AMPA/metabolismo , Adenosina Trifosfatasas/metabolismo , Encefalopatías/diagnóstico por imagen , Encefalopatías/patología , Proteínas Portadoras/metabolismo , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Homocigoto , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Mitocondrias/genética , Mitocondrias/patología , Modelos Moleculares , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Consumo de Oxígeno/genética , Transporte de Proteínas/genética , ARN Mensajero/metabolismoRESUMEN
Mutations in IQSEC2 cause intellectual disability (ID), which is often accompanied by seizures and autism. A number of studies have shown that IQSEC2 is an abundant protein in excitatory synapses and plays an important role in neuronal development as well as synaptic plasticity. Here, we review neuronal IQSEC2 signaling with emphasis on those aspects likely to be involved in autism. IQSEC2 is normally bound to N-methyl-D-aspartate (NMDA)-type glutamate receptors via post synaptic density protein 95 (PSD-95). Activation of NMDA receptors results in calcium ion influx and binding to calmodulin present on the IQSEC2 IQ domain. Calcium/calmodulin induces a conformational change in IQSEC2 leading to activation of the SEC7 catalytic domain. GTP is exchanged for GDP on ADP ribosylation factor 6 (ARF6). Activated ARF6 promotes downregulation of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors through a c-jun N terminal kinase (JNK)-mediated pathway. NMDA receptors, AMPA receptors, and PSD-95 are all known to be adversely affected in autism. An IQSEC2 transgenic mouse carrying a constitutively active mutation (A350V) shows autistic features and reduced levels of surface AMPA receptor subunit GluA2. Sec7 activity and AMPA receptor recycling are presented as two targets, which may respond to drug treatment in IQSEC2-associated ID and autism.
Asunto(s)
Trastorno Autístico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Discapacidad Intelectual/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Trastorno Autístico/tratamiento farmacológico , Trastorno Autístico/genética , Factores de Intercambio de Guanina Nucleótido/análisis , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/genética , Terapia Molecular Dirigida , Mutación/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The majority of ER-targeted tail-anchored (TA) proteins are inserted into membranes by the Guided Entry of Tail-anchored protein (GET) system. Disruption of this system causes a subset of TA proteins to mislocalize to mitochondria. We show that the AAA+ ATPase Msp1 limits the accumulation of mislocalized TA proteins on mitochondria. Deletion of MSP1 causes the Pex15 and Gos1 TA proteins to accumulate on mitochondria when the GET system is impaired. Likely as a result of failing to extract mislocalized TA proteins, yeast with combined mutation of the MSP1 gene and the GET system exhibit strong synergistic growth defects and severe mitochondrial damage, including loss of mitochondrial DNA and protein and aberrant mitochondrial morphology. Like yeast Msp1, human ATAD1 limits the mitochondrial mislocalization of PEX26 and GOS28, orthologs of Pex15 and Gos1, respectively. GOS28 protein level is also increased in ATAD1(-/-) mouse tissues. Therefore, we propose that yeast Msp1 and mammalian ATAD1 are conserved members of the mitochondrial protein quality control system that might promote the extraction and degradation of mislocalized TA proteins to maintain mitochondrial integrity.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Ligadas a Lípidos/metabolismo , Mitocondrias/fisiología , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Células Hep G2 , Humanos , Immunoblotting , Inmunoprecipitación , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Fluorescente , Mitocondrias/metabolismo , Consumo de Oxígeno/fisiología , Fosfoproteínas/metabolismo , Plásmidos/genética , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteínas SNARE/metabolismo , Saccharomyces cerevisiaeRESUMEN
Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated "parthanatos" in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD(+) and energetic collapse, which have been thought to be caused by the consumption of cellular NAD(+) by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD(+) depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD(+) depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1-mediated mitochondrial dysfunction. Depleting neurons of NAD(+) with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.
Asunto(s)
Corteza Cerebral/enzimología , Glucólisis/fisiología , Mitocondrias/enzimología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Acrilamidas/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucólisis/efectos de los fármacos , Hexoquinasa/metabolismo , Metilnitronitrosoguanidina/farmacología , Ratones , NAD/metabolismo , Neuronas/citología , Piperidinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10(-4)), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.
Asunto(s)
Gangliósido G(M2)/metabolismo , Gangliósidos/metabolismo , Receptores AMPA/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Neuronas/metabolismo , Unión Proteica , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida/metabolismoAsunto(s)
Encefalopatías , Síndrome de la Persona Rígida , Humanos , Lactante , Rigidez Muscular , Mutación , Receptores AMPARESUMEN
The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.
Asunto(s)
Aminoácidos , Serina-Treonina Quinasas TOR , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Aminoácidos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Fosforilación , Proteína Reguladora Asociada a mTOR/metabolismo , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.
Asunto(s)
Citoplasma/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Factor de Conjugación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Cisteína/química , Dimerización , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores del Factor de Conjugación/química , Receptores del Factor de Conjugación/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with â¼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.
Asunto(s)
Dihidroxifenilalanina/química , Ácido Peryódico/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Unión Competitiva , Reactivos de Enlaces Cruzados/química , Cinética , Ligandos , Espectrometría de Masas/métodos , Mitógenos/química , Mutagénesis Sitio-Dirigida , Péptidos/química , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Accumulation of the parkin-interacting substrate (PARIS; ZNF746), due to inactivation of parkin, contributes to Parkinson's disease (PD) through repression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; PPARGC1A) activity. Here, we identify farnesol as an inhibitor of PARIS. Farnesol promoted the farnesylation of PARIS, preventing its repression of PGC-1α via decreasing PARIS occupancy on the PPARGC1A promoter. Farnesol prevented dopaminergic neuronal loss and behavioral deficits via farnesylation of PARIS in PARIS transgenic mice, ventral midbrain transduction of AAV-PARIS, adult conditional parkin KO mice, and the α-synuclein preformed fibril model of sporadic PD. PARIS farnesylation is decreased in the substantia nigra of patients with PD, suggesting that reduced farnesylation of PARIS may play a role in PD. Thus, farnesol may be beneficial in the treatment of PD by enhancing the farnesylation of PARIS and restoring PGC-1α activity.
Asunto(s)
Enfermedad de Parkinson , Animales , Dopamina , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Prenilación , Proteínas Represoras/metabolismo , Sustancia Negra/metabolismoRESUMEN
The regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking affects multiple brain functions, such as learning and memory. We have previously shown that Thorase plays an important role in the internalization of AMPARs from the synaptic membrane. Here, we show that N-methyl-d-aspartate receptor (NMDAR) activation leads to increased S-nitrosylation of Thorase and N-ethylmaleimide-sensitive factor (NSF). S-nitrosylation of Thorase stabilizes Thorase-AMPAR complexes and enhances the internalization of AMPAR and interaction with protein-interacting C kinase 1 (PICK1). S-nitrosylated NSF is dependent on the S-nitrosylation of Thorase via trans-nitrosylation, which modulates the surface insertion of AMPARs. In the presence of the S-nitrosylation-deficient C137L Thorase mutant, AMPAR trafficking, long-term potentiation, and long-term depression are impaired. Overall, our data suggest that both S-nitrosylation and interactions of Thorase and NSF/PICK1 are required to modulate AMPAR-mediated synaptic plasticity. This study provides critical information that elucidates the mechanism underlying Thorase and NSF-mediated trafficking of AMPAR complexes.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Membrana Celular/metabolismo , Proteínas Sensibles a N-Etilmaleimida/metabolismo , Receptores AMPA/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Cisteína/metabolismo , Endocitosis/efectos de los fármacos , Glutatión/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , N-Metilaspartato/farmacología , Plasticidad Neuronal , Óxido Nítrico/metabolismo , Nitrosación , Unión Proteica , Multimerización de Proteína , Transporte de Proteínas , S-Nitrosoglutatión/metabolismoRESUMEN
The interaction between a 3,4-dihydroxyphenylalanine (DOPA) labeled analogue of the tridecapeptide alpha-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized alpha-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine(7)([Lys(7)(BioACA),Nle(12),DOPA(13)]alpha-factor; Bio-DOPA-alpha-factor) was used for cross-linking into Ste2p. The biological activity of Bio-DOPA-alpha-factor was about one-third that of native alpha-factor as determined by growth arrest assay and exhibited about a 10-fold lower binding affinity to Ste2p. Bio-DOPA-alpha-factor cross-linked into Ste2p as demonstrated by Western blot analysis using a neutravidin-HRP conjugate to detect Bio-DOPA-alpha-factor. Cross-linking was inhibited by excess native alpha-factor and an alpha-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-alpha-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-alpha-factor reacted with the Phe(55)-Met(69) region of Ste2p. Cross-linking of Bio-DOPA-alpha-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position 13 of alpha-factor and residue Cys(59) of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and alpha-factor, thereby defining a specific contact point between the bound ligand and its receptor.
Asunto(s)
Dihidroxifenilalanina/química , Oligopéptidos/química , Receptores Acoplados a Proteínas G/química , Receptores del Factor de Conjugación/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Sitios de Unión/genética , Membrana Celular/química , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/química , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Immunoblotting , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Factor de Conjugación/genética , Receptores del Factor de Conjugación/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transformación Genética , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, ß-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid ß-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid ß-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Proteínas Mitocondriales/deficiencia , Fibras Musculares Esqueléticas/metabolismo , Obesidad/metabolismo , Factores de Transcripción/deficiencia , Animales , Sistemas CRISPR-Cas/genética , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/genética , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Femenino , Humanos , Insulina , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Fibras Musculares Esqueléticas/citología , Obesidad/etiología , Obesidad/genética , Oxidación-Reducción , Fosforilación Oxidativa , Consumo de Oxígeno , Factores de Transcripción/genética , Aumento de Peso , Dedos de ZincRESUMEN
We have recently described an A350V mutation in IQSEC2 associated with intellectual disability, autism and epilepsy. We sought to understand the molecular pathophysiology of this mutation with the goal of developing targets for drug intervention. We demonstrate here that the A350V mutation results in interference with the binding of apocalmodulin to the IQ domain of IQSEC2. We further demonstrate that this mutation results in constitutive activation of the guanine nucleotide exchange factor (GEF) activity of IQSEC2 resulting in increased production of the active form of Arf6. In a CRISPR generated mouse model of the A350V IQSEC2 mutation, we demonstrate that the surface expression of GluA2 AMPA receptors in mouse hippocampal tissue was significantly reduced in A350V IQSEC2 mutant mice compared to wild type IQSEC2 mice and that there is a significant reduction in basal synaptic transmission in the hippocampus of A350V IQSEC2 mice compared to wild type IQSEC2 mice. Finally, the A350V IQSEC2 mice demonstrated increased activity, abnormal social behavior and learning as compared to wild type IQSEC2 mice. These findings suggest a model of how the A350V mutation in IQSEC2 may mediate disease with implications for targets for drug therapy. These studies provide a paradigm for a personalized approach to precision therapy for a disease that heretofore has no therapy.
RESUMEN
OBJECTIVE: ATAD1 encodes Thorase, a mediator of α-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptor recycling; in this work, we characterized the phenotype resulting from ATAD1 mutations and developed a targeted therapy in both mice and humans. METHODS: Using exome sequencing, we identified a novel ATAD1 mutation (p.E276X) as the etiology of a devastating neurologic disorder characterized by hypertonia, seizures, and death in a consanguineous family. We postulated that pathogenesis was a result of excessive AMPA receptor activity and designed a targeted therapeutic approach using perampanel, an AMPA-receptor antagonist. RESULTS: Perampanel therapy in ATAD1 knockout mice reversed behavioral defects, normalized brain MRI abnormalities, prevented seizures, and prolonged survival. The ATAD1 patients treated with perampanel showed improvement in hypertonicity and resolution of seizures. CONCLUSIONS: This work demonstrates that identification of novel monogenic neurologic disorders and observation of response to targeted therapeutics can provide important insights into human nervous system functioning.
RESUMEN
The AAA+ adenosine triphosphatase (ATPase) Thorase plays a critical role in controlling synaptic plasticity by regulating the expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Bidirectional sequencing of exons of ATAD1, the gene encoding Thorase, in a cohort of patients with schizophrenia and healthy controls revealed rare Thorase variants. These variants caused defects in glutamatergic signaling by impairing AMPAR internalization and recycling in mouse primary cortical neurons. This contributed to increased surface expression of the AMPAR subunit GluA2 and enhanced synaptic transmission. Heterozygous Thorase-deficient mice engineered to express these Thorase variants showed altered synaptic transmission and several behavioral deficits compared to heterozygous Thorase-deficient mice expressing wild-type Thorase. These behavioral impairments were rescued by the competitive AMPAR antagonist Perampanel, a U.S. Food and Drug Administration-approved drug. These findings suggest that Perampanel may be useful for treating disorders involving compromised AMPAR-mediated glutamatergic neurotransmission.