RESUMO
Calcineurin B homologous protein 3 (CHP3) is an EF-hand Ca2+-binding protein involved in regulation of cancerogenesis, cardiac hypertrophy, and neuronal development through interactions with sodium/proton exchangers (NHEs) and signalling proteins. While the importance of Ca2+ binding and myristoylation for CHP3 function has been recognized, the underlying molecular mechanism remained elusive. In this study, we demonstrate that Ca2+ binding and myristoylation independently affect the conformation and functions of human CHP3. Ca2+ binding increased local flexibility and hydrophobicity of CHP3 indicative of an open conformation. The Ca2+-bound CHP3 exhibited a higher affinity for NHE1 and associated stronger with lipid membranes compared to the Mg2+-bound CHP3, which adopted a closed conformation. Myristoylation enhanced the local flexibility of CHP3 and decreased its affinity to NHE1 independently of the bound ion, but did not affect its binding to lipid membranes. The data exclude the proposed Ca2+-myristoyl switch for CHP3. Instead, a Ca2+-independent exposure of the myristoyl moiety is induced by binding of the target peptide to CHP3 enhancing its association to lipid membranes. We name this novel regulatory mechanism 'target-myristoyl switch'. Collectively, the interplay of Ca2+ binding, myristoylation, and target binding allows for a context-specific regulation of CHP3 functions.
Assuntos
Calcineurina , Proteínas de Ligação ao Cálcio , Humanos , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Conformação Molecular , Prótons , Lipídeos , Cálcio/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.
Assuntos
Cistos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Receptores Acoplados a Proteínas G/genética , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neurônio-Glia/genética , Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Proteínas de Ciclo Celular/genética , Canais de Cloreto/genética , Cistos/metabolismo , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/metabolismo , Transporte Proteico , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.
Assuntos
Basigina/metabolismo , Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Subunidades Proteicas/metabolismoRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.
Assuntos
Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Encefalopatias/patologia , Canais de Cloro CLC-2 , Canais de Cálcio Tipo L/genética , Canais de Cloreto , Cistos/genética , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Transporte Proteico/genéticaRESUMO
Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.
Assuntos
Antígenos CD13/metabolismo , Mastócitos/fisiologia , Anafilaxia , Animais , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/genética , Proliferação de Células , Dinitrofenóis/imunologia , Regulação da Expressão Gênica/fisiologia , Leucina/análogos & derivados , Leucina/farmacologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgE/genética , Receptores de IgE/metabolismo , Albumina Sérica/imunologiaRESUMO
Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl(-) channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.
Assuntos
Canais de Cloreto/fisiologia , Neuroglia/metabolismo , Animais , Biofísica , Canais de Cloro CLC-2 , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/ultraestrutura , Conexinas/metabolismo , Estimulação Elétrica , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Espectrometria de Massas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Microinjeções/métodos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutação/genética , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Cadeias Leves de Miosina/genética , Neuroglia/ultraestrutura , Oócitos , Técnicas de Patch-Clamp , Transporte Proteico/genética , Ratos , Transfecção , XenopusRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly.
Assuntos
Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Deficiência Intelectual/genética , Megalencefalia/genética , Mutação , Proteínas/genética , Sequência de Aminoácidos , Animais , Transtorno Autístico/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cistos/genética , Cistos/metabolismo , Genes Dominantes , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Megalencefalia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key modulators of neuronal activity by providing the depolarizing cation current I(h) involved in rhythmogenesis, dendritic integration, and synaptic transmission. These tasks critically depend on the availability of HCN channels, which is dynamically regulated by intracellular cAMP; the range of this regulation, however, largely differs among neurons in the mammalian brain. Using affinity purification and high-resolution mass spectrometry, we identify the PEX5R/Trip8b protein as the beta subunit of HCN channels in the mammalian brain. Coassembly of PEX5R/Trip8b affects HCN channel gating in a subtype-dependent and mode-specific way: activation of HCN2 and HCN4 by cAMP is largely impaired, while gating by phosphoinositides and basal voltage-dependence remain unaffected. De novo expression of PEX5R/Trip8b in cardiomyocytes abolishes beta-adrenergic stimulation of HCN channels. These results demonstrate that PEX5R/Trip8b is an intrinsic auxiliary subunit of brain HCN channels and establish HCN-PEX5R/Trip8b coassembly as a mechanism to control the channels' responsiveness to cyclic nucleotide signaling.
Assuntos
AMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Canais de Potássio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Antagonistas de Receptores Adrenérgicos beta 1 , Animais , Encéfalo/ultraestrutura , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Ativação do Canal Iônico/genética , Isoproterenol/farmacologia , Espectrometria de Massas/métodos , Potenciais da Membrana/genética , Proteínas de Membrana/genética , Microinjeções/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oócitos , Técnicas de Patch-Clamp/métodos , Peroxinas , Canais de Potássio/genética , Multimerização Proteica/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Transdução Genética/métodos , XenopusRESUMO
The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.
Assuntos
Encéfalo/metabolismo , Canal de Potássio Kv1.1/fisiologia , Canal de Potássio Kv1.2/fisiologia , Inibição Neural/fisiologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting/métodos , Encéfalo/citologia , Química Encefálica , Membrana Celular/metabolismo , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas/métodos , Potenciais da Membrana/fisiologia , Mutagênese/fisiologia , Mutação , Oócitos , Técnicas de Patch-Clamp/métodos , Conformação Proteica , Ratos , Alinhamento de Sequência , Coloração pela Prata/métodos , Transfecção/métodos , XenopusRESUMO
Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.