RESUMO
BACKGROUND AND OBJECTIVES: The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. RESULTS: Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. DISCUSSION: IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.
Assuntos
Autoanticorpos , Síndrome de Guillain-Barré , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica , Idoso , Animais , Humanos , Camundongos , Pessoa de Meia-Idade , Ratos , Proteínas ADAM , Autoanticorpos/sangue , Autoanticorpos/química , Síndrome de Guillain-Barré/diagnóstico , Células HEK293 , Imunoglobulina G , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/patologiaRESUMO
Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.
Assuntos
Cisteína , Monofenol Mono-Oxigenase , Humanos , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Lipoilação , Aciltransferases/metabolismo , Melaninas/metabolismo , Melanócitos/metabolismoRESUMO
Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.
Assuntos
Cerebelo , Canais Iônicos , Animais , Canais Iônicos/metabolismo , CamundongosRESUMO
Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.
Assuntos
Proteínas ADAM , Encefalopatias , Epilepsia Resistente a Medicamentos , Proteínas do Tecido Nervoso , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Atrofia , Encefalopatias/genética , Proteína 4 Homóloga a Disks-Large , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Optogenetics has become an indispensable tool for investigating brain functions. Although non-human primates are particularly useful models for understanding the functions and dysfunctions of the human brain, application of optogenetics to non-human primates is still limited. In the present study, we generate an effective adeno-associated viral vector serotype DJ to express channelrhodopsin-2 (ChR2) under the control of a strong ubiquitous CAG promoter and inject into the somatotopically identified forelimb region of the primary motor cortex in macaque monkeys. ChR2 is strongly expressed around the injection sites, and optogenetic intracortical microstimulation (oICMS) through a homemade optrode induces prominent cortical activity: Even single-pulse, short-duration oICMS evokes long-lasting repetitive firings of cortical neurons. In addition, oICMS elicits distinct forelimb movements and muscle activity, which are comparable to those elicited by conventional electrical ICMS. The present study removes obstacles to optogenetic manipulation of neuronal activity and behaviors in non-human primates.
Assuntos
Membro Anterior/fisiologia , Córtex Motor/fisiologia , Movimento/fisiologia , Optogenética , Animais , Channelrhodopsins/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Macaca , Neurônios/fisiologia , Estimulação FísicaRESUMO
Tricellular tight junctions (tTJs) create paracellular barriers at tricellular contacts (TCs), where the vertices of three polygonal epithelial cells meet. tTJs are marked by the enrichment of two types of membrane proteins, tricellulin and angulin family proteins. However, how TC geometry is recognized for tTJ formation remains unknown. In the present study, we examined the molecular mechanism for the assembly of angulin-1 at the TCs. We found that clusters of cysteine residues in the juxtamembrane region within the cytoplasmic domain of angulin-1 are highly palmitoylated. Mutagenesis analyses of the cysteine residues in this region revealed that palmitoylation is essential for localization of angulin-1 at TCs. Consistently, suppression of Asp-His-His-Cys motif-containing palmitoyltransferases expressed in EpH4 cells significantly impaired the TC localization of angulin-1. Cholesterol depletion from the plasma membrane of cultured epithelial cells hampered the localization of angulin-1 at TCs, suggesting the existence of a lipid membrane microdomain at TCs that attracts highly palmitoylated angulin-1. Furthermore, the extracellular domain of angulin-1 was also required for its TC localization, irrespective of the intracellular palmitoylation. Taken together, our findings suggest that both angulin-1's extracellular domain and palmitoylation of its cytoplasmic region are required for its assembly at TCs.
Assuntos
Colesterol/genética , Lipoilação/genética , Microdomínios da Membrana/genética , Receptores de Lipoproteínas/genética , Comunicação Celular/genética , Colesterol/metabolismo , Cisteína/química , Cisteína/genética , Células Epiteliais/metabolismo , Humanos , Junções Intercelulares/genética , Proteína 2 com Domínio MARVEL , Microdomínios da Membrana/química , Domínios Proteicos/genética , Processamento de Proteína Pós-Traducional/genética , Receptores de Lipoproteínas/química , Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediated encephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype distribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from the cerebrospinal fluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereas reactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had undergone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with its receptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining, non-ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excitability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. INTERPRETATION: Our data show that the patients' intrathecal B-cell autoimmune response is dominated by LGI1 antibodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation. Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody repertoire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides are very distinct entities. Ann Neurol 2020;87:405-418.
Assuntos
Anticorpos Monoclonais/farmacologia , Autoanticorpos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Neurônios/fisiologia , Proteínas ADAM/efeitos dos fármacos , Idoso , Animais , Anticorpos Monoclonais/líquido cefalorraquidiano , Autoanticorpos/líquido cefalorraquidiano , Região CA3 Hipocampal/fisiologia , Células Cultivadas , Encefalite/líquido cefalorraquidiano , Encefalite/imunologia , Feminino , Doença de Hashimoto/líquido cefalorraquidiano , Doença de Hashimoto/imunologia , Humanos , Isotipos de Imunoglobulinas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/efeitos dos fármacos , Ratos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Dickkopf1 (DKK1) was originally identified as an antagonist of Wnt signaling that binds to and induces the clathrin-mediated endocytosis of the Wnt coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). DKK1 also binds to cytoskeleton-associated protein 4 (CKAP4), which was originally identified as an endoplasmic reticulum (ER) protein but also functions at the plasma membrane as a receptor for various ligands. The DKK1-CKAP4 pathway is activated in several human cancers and promotes cell proliferation by activating signaling through the kinases PI3K and AKT. We found that both CKAP4 and LRP6 primarily localized to detergent-resistant membrane (DRM) fractions of the plasma membrane in a palmitoylation-dependent manner and that palmitoylation of CKAP4 was required for it to promote cell proliferation. DKK1 induced the depalmitoylation of both CKAP4 and LRP6 by acylprotein thioesterases (APTs), resulting in their translocation to the non-DRM fractions. Moreover, DKK1-dependent depalmitoylation of both receptors required activation of the PI3K-AKT pathway. DKK1 simultaneously bound CKAP4 and LRP6, resulting in the formation of a ternary complex. LRP5/6 knockdown decreased DKK1-dependent AKT activation and cancer cell proliferation through CKAP4, whereas CKAP4 knockdown did not affect DKK1-dependent inhibition of Wnt signaling through LRP5/6. These results indicate that the palmitoylation states of CKAP4 and LRP6 play important roles in their signaling and that LRP5/6 enhance DKK1-CKAP4 signaling.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Endocitose , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lipoilação , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Palmitoil-CoA Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/genéticaRESUMO
Using a cell-based assay monitoring differential protein transport in the secretory pathway coupled to high-content screening, we have identified three molecules that specifically reduce the delivery of the major co-receptor for HIV-1, CCR5, to the plasma membrane. They have no effect on the closely related receptors CCR1 and CXCR4. These molecules are also potent in primary macrophages as they markedly decrease HIV entry. At the molecular level, two of these molecules inhibit the critical palmitoylation of CCR5 and thereby block CCR5 in the early secretory pathway. Our results open a clear therapeutics avenue based on trafficking control and demonstrate that preventing HIV infection can be performed at the level of its receptor delivery.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Transporte Proteico/fisiologia , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Receptores CCR1/metabolismo , Receptores CXCR4/metabolismo , Via Secretória/fisiologiaRESUMO
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 , Imageamento por Ressonância Magnética/veterinária , Prednisolona/uso terapêutico , Convulsões/tratamento farmacológico , Estado Epiléptico/tratamento farmacológicoRESUMO
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 5â¯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.
Assuntos
Doenças Autoimunes do Sistema Nervoso/imunologia , Ataxia Cerebelar/imunologia , Receptores de Glutamato Metabotrópico/imunologia , Atrofia/patologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes do Sistema Nervoso/tratamento farmacológico , Doenças Autoimunes do Sistema Nervoso/patologia , Ataxia Cerebelar/tratamento farmacológico , Ataxia Cerebelar/patologia , Feminino , Seguimentos , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Imunossupressores/uso terapêutico , Pessoa de Meia-IdadeRESUMO
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/genéticaRESUMO
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 , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Transmissão SinápticaRESUMO
MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Células 3T3 , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Camundongos , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Ligação Proteica , Multimerização Proteica , Transporte ProteicoRESUMO
Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the ß3 subunit of the GABA(A) receptor. The ß3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the ß3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies.
Assuntos
Autoanticorpos/sangue , Encefalopatias/sangue , Encefalopatias/imunologia , Encéfalo/patologia , Doença de Hashimoto/sangue , Doença de Hashimoto/imunologia , Receptores de GABA-A/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Encéfalo/metabolismo , Encefalopatias/complicações , Encefalopatias/patologia , Células Cultivadas , Chlorocebus aethiops , Transtornos Cognitivos/etiologia , Encefalite , Feminino , Doença de Hashimoto/complicações , Doença de Hashimoto/patologia , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotransmissores/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas/imunologia , RatosRESUMO
The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.
Assuntos
Glicoproteínas/química , Glicoproteínas/metabolismo , Bainha de Mielina/metabolismo , Filogenia , Proteínas ADAM/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Axônios/metabolismo , Sequência Conservada , Teste de Complementação Genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Sistema Nervoso Periférico/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures. We comprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain of ADAM22 was sufficient to reduce synaptic AMPA receptors in rat hippocampal neurons and that levels of AMPA receptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces the AMPA receptor function, causing epileptic disorders. These results suggest that by finely regulating the synaptic AMPA receptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life.
Assuntos
Proteínas ADAM/metabolismo , Autoanticorpos/sangue , Epilepsia/sangue , Encefalite Límbica/sangue , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Receptores de AMPA/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células COS , Criança , Pré-Escolar , Chlorocebus aethiops , Epilepsia/diagnóstico , Feminino , Células HEK293 , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular , Encefalite Límbica/diagnóstico , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Ligação Proteica/fisiologia , Ratos , Adulto JovemRESUMO
Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca(2+)/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.
Assuntos
Aciltransferases/metabolismo , Endossomos/metabolismo , Lipoilação/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Software , Proteínas rab5 de Ligação ao GTP/metabolismo , Aciltransferases/genética , Animais , Cisteína/genética , Cisteína/metabolismo , Endossomos/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Ratos , Especificidade por Substrato , Sinapses/genética , Sinapses/metabolismo , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
Leucine-rich glioma inactivated 1 (Lgi1) is a secreted synaptic protein that organizes a transsynaptic protein complex throughout the brain. Mutations in the Lgi1 gene have been found in patients with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although a large number of studies have focused on the expression and function of Lgi1 in the postnatal brain, information regarding its functions and distribution during development remains sparse. Here we report that Lgi1 mRNA is preferentially expressed in the caudal ganglionic eminence (CGE) of the early embryonic telencephalon, and LGI1 protein is unexpectedly localized in the nucleus of dissociated CGE neurons. Using bioinformatics analysis, we found that LGI1 contains a putative nuclear localization signal (NLS) in its leucine-rich repeat C-terminal domain. Furthermore, we show that the transient expression of Lgi1 in CGE neurons resulted in nuclear translocation of the LGI1 protein, and a mutation in the NLS led to the retention of LGI1 in the cytoplasm. We also confirmed that the NLS sequence of LGI1 had the ability to mediate the nuclear localization by using the NLS-containing fusion protein. Interestingly, when Lgi1 was expressed in neurons obtained from the medial ganglionic eminence or cerebral cortex, almost no nuclear localization of LGI1 was observed. These results raise the possibility of a novel role of Lgi1 within embryonic neurons through nuclear translocation and may provide insight into its potential effects on the development of the central nervous system and ADLTE pathogenesis.