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1.
Methods Mol Biol ; 2009: 83-98, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31152397

RESUMO

Palmitoylation is a reversible posttranslational lipid modification of proteins involved in a wide range of cellular functions. More than a thousand proteins are estimated to be palmitoylated. In neurons, PSD-95, a major postsynaptic scaffold protein, requires palmitoylation for its specific accumulation at the synapse and dynamically cycles between palmitoylated and depalmitoylated states. Although palmitoylating enzymes of PSD-95 have been well characterized, little is known about the depalmitoylating enzymes (e.g., thioesterases for palmitoylated PSD-95). An elegant pharmacological analysis has suggested that subsets of α/ß hydrolase domain (ABHD)-containing proteins of the metabolic serine hydrolase superfamily involve thioesterases for palmitoylated proteins. Here, we describe a systematic method to screen the ABHD serine hydrolase genes, which unveiled ABHD17 as the depalmitoylating enzyme for PSD-95. Furthermore, we introduce the acyl-PEGyl exchange gel-shift (APEGS) method that enables quantification of palmitoylation levels/stoichiometries on proteins in various biological samples and can be used to monitor the dynamic depalmitoylation process of proteins.

2.
J Vet Intern Med ; 33(3): 1440-1445, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942925

RESUMO

A 7-year-old neutered female domestic shorthaired cat born in Poland and then moved to Japan presented to the local clinic with recent onset of convulsive cluster seizures and status epilepticus. Magnetic resonance imaging revealed bilateral swelling of the hippocampus with T2 hyperintensity and contrast enhancing image, suggesting hippocampal necrosis. The cat completely recovered after treatment with antiepileptic drugs (AED) and administration of prednisolone (1 mg/kg PO q24h for 4 days and tapered). However, cluster seizures reoccurred and developed into status epilepticus despite increasing doses of AED. Although the convulsions were resolved by other AEDs, stupor and renal failure developed, and the cat was euthanized. Pathological findings were consistent with hippocampal necrosis. Immunological analysis for leucine-rich glioma inactivated 1 (LGI1) autoantibodies was negative, but antibodies against DCC (deleted in colorectal carcinoma) known as netrin-1 receptor were found. This report describes a case of feline autoimmune limbic encephalitis and hippocampal necrosis that were presumably associated with DCC autoantibodies.


Assuntos
Doenças Autoimunes/veterinária , Doenças do Gato/imunologia , Hipocampo/patologia , Encefalite Límbica/veterinária , Necrose/veterinária , Receptores de Netrina/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Anticonvulsivantes/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Gatos , Feminino , Hipocampo/diagnóstico por imagem , Encefalite Límbica/tratamento farmacológico , Encefalite Límbica/imunologia , Imagem por Ressonância Magnética/veterinária , Prednisolona/uso terapêutico , Convulsões/tratamento farmacológico , Estado Epiléptico/tratamento farmacológico
3.
Nat Commun ; 9(1): 1546, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29670100

RESUMO

Epilepsy is a common brain disorder throughout history. Epilepsy-related ligand-receptor complex, LGI1-ADAM22, regulates synaptic transmission and has emerged as a determinant of brain excitability, as their mutations and acquired LGI1 autoantibodies cause epileptic disorders in human. Here, we report the crystal structure of human LGI1-ADAM22 complex, revealing a 2:2 heterotetrameric assembly. The hydrophobic pocket of the C-terminal epitempin-repeat (EPTP) domain of LGI1 binds to the metalloprotease-like domain of ADAM22. The N-terminal leucine-rich repeat and EPTP domains of LGI1 mediate the intermolecular LGI1-LGI1 interaction. A pathogenic R474Q mutation of LGI1, which does not exceptionally affect either the secretion or the ADAM22 binding, is located in the LGI1-LGI1 interface and disrupts the higher-order assembly of the LGI1-ADAM22 complex in vitro and in a mouse model for familial epilepsy. These studies support the notion that the LGI1-ADAM22 complex functions as the trans-synaptic machinery for precise synaptic transmission.

4.
J Neuroimmunol ; 319: 63-67, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29685291

RESUMO

The clinical features of cerebellar ataxia associated with anti-metabotropic glutamate receptor 1 (mGluR1) autoantibodies, a rare autoimmune-mediated cerebellar ataxia, remain to be elucidated. Here, we describe a patient with non-paraneoplastic cerebellar ataxia associated with anti-mGluR1 autoantibodies, who was followed up over 5 years. She presented with relapses and remissions of subacute progressive cerebellar ataxia that were responsive to immunotherapy. Although serum anti-mGluR1 autoantibodies were continuously detected and cerebellar atrophy gradually progressed, repeated intravenous immunoglobulin therapy and oral immunosuppressants ensured cerebellar ataxia remained at almost the same level during the observation period.

5.
Curr Opin Neurobiol ; 48: 1-8, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28829986

RESUMO

Autoimmune encephalitis presenting with amnesia, seizures, and psychosis is highly topical in basic and clinical neuroscience. Recent studies have identified numerous associated autoantibodies, targeting cell-surface synaptic proteins including neurotransmitter receptors (e.g. NMDA receptors (NMDARs)) and a secreted protein, LGI1. In vitro and in vivo analyses of the influence of the autoantibodies have begun to clarify their causal roles. Of particular interest is the generation of recombinant monoclonal antibodies from patients' B cells with anti-NMDAR encephalitis. Patient monoclonal antibodies could be useful to reveal their direct, detailed pathogenicity. Such identification and characterization of autoantibodies could create new categories of neurological diseases and promote the understanding of patho-physiologic roles of target proteins in human brain function.

6.
Neuron ; 94(4): 809-825.e7, 2017 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-28521134

RESUMO

Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/ß Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.


Assuntos
Potenciais de Ação , Axônios/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Ácido Palmítico/metabolismo , Vesículas Secretórias/metabolismo , Animais , Células COS , Cercopithecus aethiops , Hipocampo/citologia , Técnicas In Vitro , Lipoilação , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar
7.
J Biochem ; 162(4): 295-302, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28449070

RESUMO

Neuronal synapse formation is regulated by pre- and postsynaptic cell adhesion molecules. Presynaptic neurexins (NRXNs) and receptor protein tyrosine phosphatases (RPTPs; PTPδ, PTPσ and LAR in mammals) can induce postsynaptic differentiation through the interaction with various postsynaptic cell adhesion molecules. Here, we developed a novel in situ screening method to identify postsynaptic membranous proteins involved in synaptogenesis. Magnetic beads coated with the extracellular domains of NRXN1ß(-S4) and PTPδ-A6 variants preferentially induced excitatory postsynaptic differentiation on the beads' surface when co-cultured with cortical neurons. After inducing postsynaptic sites on these beads, protein complexes including NRXN1ß(-S4)/PTPδ-A6 and their ligands on the neuronal membrane were chemically cross-linked and purified using a magnetic separator. Liquid chromatography-tandem mass spectrometry analysis of the complexes revealed two types of postsynaptic ligands for NRXN1ß(-S4) and PTPδ-A6, one has an activity to induce presynaptic differentiation in a trans manner, whereas the other has no such activity. These results suggest that synapse formation is regulated by the interplay between presynaptic NRXN/PTPδ and their postsynaptic ligands with functionally different impacts on pre- and postsynaptic differentiation. Thus, our in situ screening method for identifying synapse-organizing complexes will help to understand the molecular basis for elaborate neuronal networks.


Assuntos
Moléculas de Adesão Celular/análise , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Cromatografia Líquida , Técnicas de Cocultura , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Espectrometria de Massas em Tandem
8.
Curr Opin Neurobiol ; 45: 1-8, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28219682

RESUMO

Throughout history, epilepsy affects about 1-2% of the population worldwide. Epilepsy can be caused by traumatic brain injury, exposure to certain toxins and drugs, and mutations of genes that often encode synaptic proteins. In addition to conventional linkage and association studies, the recent trio exome sequencing in epilepsy and proteomic analysis in autoimmune synaptopathies have accelerated identification of novel epilepsy-related proteins, most of which play critical roles in synaptic transmission. Furthermore, super-resolution microscopy analysis has revealed subsynaptic nanoscale distribution of presynaptic and postsynaptic proteins and suggests a precise trans-synaptic alignment of neurotransmitter release to receptors. Such identification and characterization of epilepsy-related synaptic proteins have been promoting the development of anti-epileptic drugs and the understanding of mechanisms of synaptic transmission.


Assuntos
Epilepsia/genética , Proteínas do Tecido Nervoso/fisiologia , Epilepsia/etiologia , Humanos , Proteômica , Transmissão Sináptica/fisiologia
9.
J Neurosci ; 37(12): 3181-3191, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213441

RESUMO

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.


Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Ligação Proteica
10.
Genes Cells ; 22(1): 94-104, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27935186

RESUMO

Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+ . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α1 and auxiliary α2 /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+ -ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+ -pumping activity.


Assuntos
Canais de Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteômica , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Glicoproteínas de Membrana/genética , Mapeamento de Interação de Proteínas/métodos , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
11.
Neurosci Res ; 116: 39-45, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27717669

RESUMO

Physiological functioning of the brain requires fine-tuned synaptic transmission, and its dysfunction causes various brain disorders such as autism, dementia, and epilepsy. It is therefore extremely important to identify and characterize key regulators of synaptic function. In particular, disease-related synaptic proteins, such as autism-related neurexin-neuroligin and psychiatric disorder-related NMDA receptor, have attracted considerable attention. Recent basic and clinical research has highlighted critical roles of a ligand-receptor complex, LGI1-ADAM22, in synaptic transmission and brain function, as mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy and autoantibodies to LGI1 cause limbic encephalitis which is characterized by memory loss and seizures. Here, we will review our current knowledge about LGI1 and ADAM22, and discuss their patho-physiological roles in synaptic transmission and synaptic disorders.


Assuntos
Proteínas ADAM/metabolismo , Encefalopatias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Transmissão Sináptica , Proteínas ADAM/genética , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Encéfalo/metabolismo , Encefalopatias/fisiopatologia , Humanos , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas/genética , Proteínas/imunologia
12.
Glia ; 65(1): 150-168, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27748972

RESUMO

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile-onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte-specific Mlc1 overexpression causes infantile-onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+ /K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+ /K+ pump. In contrast, no changes in Na+ /K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+ /K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile-onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile-onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150-168.


Assuntos
Astrócitos/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/genética , Substância Branca/metabolismo , Animais , Membrana Celular/metabolismo , Cistos/genética , Modelos Animais de Doenças , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Mutação/genética
13.
J Neurosci ; 36(24): 6431-44, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27307232

RESUMO

UNLABELLED: Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/ß-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT: Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.


Assuntos
Guanilato Quinases/metabolismo , Lipoilação/fisiologia , Proteínas de Membrana/metabolismo , Monoacilglicerol Lipases/metabolismo , Neurônios/enzimologia , Serina/análogos & derivados , Animais , Linhagem Celular Transformada , Cercopithecus aethiops , Cerebelo/metabolismo , Proteína 4 Homóloga a Disks-Large , Feminino , Guanilato Quinases/genética , Hipocampo/citologia , Hidrolases/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Monoacilglicerol Lipases/genética , Palmitatos/metabolismo , Transporte Proteico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Serina/isolamento & purificação , Serina/metabolismo , Frações Subcelulares/metabolismo , Especificidade por Substrato , Trítio/metabolismo
14.
Neurol Genet ; 2(1): e46, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27066583

RESUMO

OBJECTIVE: To identify the molecular genetic basis of a syndrome characterized by rapidly progressing cerebral atrophy, intractable seizures, and intellectual disability. METHODS: We performed exome sequencing in the proband and whole-genome single nucleotide polymorphism genotyping (copy number variant analysis) in the proband-parent trio. We used heterologous expression systems to study the functional consequences of identified mutations. RESULTS: The search for potentially deleterious recessive or de novo variants yielded compound heterozygous missense (c.1202G>A, p.Cys401Tyr) and frameshift deletion (c.2396delG, p.Ser799IlefsTer96) mutations in ADAM22, which encodes a postsynaptic receptor for LGI1. The deleterious effect of the mutations was observed in cell surface binding and immunoprecipitation assays, which revealed that both mutant proteins failed to bind to LGI1. Furthermore, immunoprecipitation assays showed that the frameshift mutant ADAM22 also did not bind to the postsynaptic scaffolding protein PSD-95. CONCLUSIONS: The mutations identified abolish the LGI1-ADAM22 ligand-receptor complex and are thus a likely primary cause of the proband's epilepsy syndrome, which is characterized by unusually rapidly progressing cortical atrophy starting at 3-4 months of age. These findings are in line with the implicated role of the LGI1-ADAM22 complex as a key player in nervous system development, specifically in functional maturation of postnatal synapses. Because the frameshift mutation affects an alternatively spliced exon with highest expression in postnatal brain, the combined effect of the mutations is likely to be hypomorphic rather than complete loss of function. This is compatible with the longer survival of the patient compared to Lgi1 (-/-) and Adam22 (-/-) mice, which develop lethal seizures during the first postnatal weeks.

15.
Curr Top Membr ; 77: 97-141, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26781831

RESUMO

Palmitoylation is an evolutionally conserved lipid modification of proteins. Dynamic and reversible palmitoylation controls a wide range of molecular and cellular properties of proteins including the protein trafficking, protein function, protein stability, and specialized membrane domain organization. However, technical difficulties in (1) detection of palmitoylated substrate proteins and (2) purification and enzymology of palmitoylating enzymes have prevented the progress in palmitoylation research, compared with that in phosphorylation research. The recent development of proteomic and chemical biology techniques has unexpectedly expanded the known complement of palmitoylated proteins in various species and tissues/cells, and revealed the unique occurrence of palmitoylated proteins in membrane-bound organelles and specific membrane compartments. Furthermore, identification and characterization of DHHC (Asp-His-His-Cys) palmitoylating enzyme-substrate pairs have contributed to elucidating the regulatory mechanisms and pathophysiological significance of protein palmitoylation. Here, we review the recent progress in protein palmitoylation at the molecular, cellular, and in vivo level and discuss how locally regulated palmitoylation machinery works for dynamic nanoscale organization of membrane domains.


Assuntos
Lipoilação , Microdomínios da Membrana/metabolismo , Animais , Humanos , Espaço Intracelular/metabolismo , Proteínas/metabolismo
16.
Proc Natl Acad Sci U S A ; 112(30): E4129-37, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26178195

RESUMO

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.


Assuntos
Proteínas ADAM/metabolismo , Regulação da Expressão Gênica , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Sinapses/fisiologia , Motivos de Aminoácidos , Animais , Encéfalo/patologia , Membrana Celular/metabolismo , Proteína 4 Homóloga a Disks-Large , Eletrodos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Transmissão Sináptica
17.
Mol Biol Cell ; 26(12): 2333-42, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25904333

RESUMO

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.


Assuntos
Adipócitos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Lipoilação , Fator 1 de Ribosilação do ADP/metabolismo , Adipócitos/enzimologia , Complexo I de Proteína do Envoltório/metabolismo , Regulação da Expressão Gênica , Humanos , Lipase/genética , Triglicerídeos/metabolismo
18.
Biochem Soc Trans ; 43(2): 199-204, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25849917

RESUMO

Precise regulation of protein assembly at specialized membrane domains is essential for diverse cellular functions including synaptic transmission. However, it is incompletely understood how protein clustering at the plasma membrane is initiated, maintained and controlled. Protein palmitoylation, a common post-translational modification, regulates protein targeting to the plasma membrane. Such modified proteins are enriched in these specialized membrane domains. In this review, we focus on palmitoylation of PSD-95, which is a major postsynaptic scaffolding protein and makes discrete postsynaptic nanodomains in a palmitoylation-dependent manner and discuss a determinant role of local palmitoylation cycles in creating highly localized hotspots at the membrane where specific proteins concentrate to organize functional domains.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoilação/genética , Proteínas de Membrana/metabolismo , Transmissão Sináptica/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Proteína 4 Homóloga a Disks-Large , Hipocampo/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Neurônios/metabolismo , Sinapses/genética , Sinapses/metabolismo
19.
J Clin Invest ; 125(4): 1497-508, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25751059

RESUMO

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.


Assuntos
Proteínas Angiogênicas/fisiologia , Guanilato Quinases/metabolismo , Hipocampo/fisiopatologia , Transtornos de Aprendizagem/genética , Proteínas de Membrana/metabolismo , Transtornos da Memória/genética , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Aprendizagem Espacial/fisiologia , Proteínas Angiogênicas/deficiência , Proteínas Angiogênicas/genética , Animais , Encéfalo/irrigação sanguínea , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases/deficiência , Guanilato Quinases/genética , Células HEK293 , Hipocampo/patologia , Humanos , Curva de Aprendizado , Transtornos de Aprendizagem/fisiopatologia , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto/fisiologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Neurônios/ultraestrutura , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transmissão Sináptica/fisiologia , Ubiquitinação
20.
Nat Med ; 21(1): 19-26, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25485908

RESUMO

Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.


Assuntos
Proteínas ADAM/metabolismo , Epilepsia do Lobo Frontal/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas/genética , Convulsões/genética , Transtornos do Sono-Vigília/genética , Proteínas ADAM/química , Proteínas ADAM/genética , Animais , Modelos Animais de Doenças , Epilepsia do Lobo Frontal/patologia , Epilepsia do Lobo Frontal/terapia , Predisposição Genética para Doença , Humanos , Camundongos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fenilbutiratos/administração & dosagem , Dobramento de Proteína/efeitos dos fármacos , Proteínas/metabolismo , Convulsões/patologia , Convulsões/terapia , Transtornos do Sono-Vigília/patologia , Transtornos do Sono-Vigília/terapia
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