RESUMO
The cell signaling molecules nitric oxide (NO) and Ca2+ regulate diverse biological processes through their closely coordinated activities directed by signaling protein complexes. However, it remains unclear how dynamically the multi-component protein assemblies behave within the signaling complexes upon the interplay between NO and Ca2+ signals. Here we demonstrate that TRPC5 channels activated by stimulation of G-protein-coupled ATP receptors mediate Ca2+ influx, that triggers NO production from endothelial NO synthase (eNOS), inducing secondary activation of TRPC5 via cysteine S-nitrosylation and eNOS in vascular endothelial cells. Mutations in the caveolin-1-binding domains of TRPC5 disrupt its association with caveolin-1 and impair Ca2+ influx and NO production, suggesting that caveolin-1 serves primarily as the scaffold for TRPC5 and eNOS to assemble into the signal complex. Interestingly, during ATP receptor activation, eNOS is dissociated from caveolin-1 and in turn directly associates with TRPC5, which accumulates at the plasma membrane dependently on Ca2+ influx and calmodulin (CaM). This protein reassembly likely results in a relief of eNOS from the inhibitory action of caveolin-1 and an enhanced TRPC5 S-nitrosylation by eNOS localized in the proximity, thereby facilitating the secondary activation of Ca2+ influx and NO production. In isolated rat aorta, vasodilation induced by acetylcholine was significantly suppressed by the TRPC5 inhibitor AC1903. Thus, our study provides evidence that dynamic remodeling of the protein assemblies among TRPC5, eNOS, caveolin-1, and CaM determines the ensemble of Ca2+ mobilization and NO production in vascular endothelial cells.
RESUMO
Commensal bacteria affect many aspects of host physiology. In this study, we focused on the role of commensal bacteria in the thermoregulatory behavior of Drosophila melanogaster. We demonstrated that the elimination of commensal bacteria caused an increase in the preferred temperature of Drosophila third-instar larvae without affecting the activity of transient receptor potential ankyrin 1 (TRPA1)-expressing thermosensitive neurons. We isolated eight bacterial strains from the gut and culture medium of conventionally reared larvae and found that the preferred temperature of the larvae was decreased by mono-association with Lactobacillus plantarum or Corynebacterium nuruki. Mono-association with these bacteria did not affect the indices of energy metabolism such as ATP and glucose levels of larvae, which are closely linked to thermoregulation in animals. Thus, we show a novel role for commensal bacteria in host thermoregulation and identify two bacterial species that affect thermoregulatory behavior in Drosophila.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Bactérias , Regulação da Temperatura Corporal , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Larva/fisiologia , SimbioseRESUMO
α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Distroglicanas/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Membrana Basal/efeitos dos fármacos , Membrana Basal/virologia , Líquidos Corporais/efeitos dos fármacos , Líquidos Corporais/virologia , Linhagem Celular , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Inflamação/virologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Carga Viral/métodosRESUMO
RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.
Assuntos
Peptídeo Hidrolases/metabolismo , Fosfolipídeos/biossíntese , Picornaviridae/enzimologia , Proteínas Virais/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Brefeldina A/farmacologia , Membrana Celular/ultraestrutura , Dactinomicina/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Biogênese de Organelas , Fosfatos de Fosfatidilinositol/metabolismo , Poliovirus/enzimologia , Inibidores da Síntese de Proteínas/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologiaRESUMO
PURPOSE: To determine the changes in the cone-driven functions in patients with age-related macular degeneration (AMD) treated with intravitreal aflibercept. METHODS: We studied 44 eyes of 44 patients diagnosed with AMD whose mean age was 75 years. The contralateral unaffected eyes served as controls. All patients were initially treated with 3 consecutive monthly intravitreal aflibercept injections and thereafter with bimonthly injections for 12 months. Full-field cone electroretinograms (cone ERGs) were recorded at the baseline and at 3, 6, and 12 months after beginning the intravitreal aflibercept injections. The cone ERGs were elicited by red stimuli on a blue background. The focal macular ERGs (fmERGs) were elicited by 15 degrees white stimulus spot centered on the fovea. The amplitudes of the a- and b-waves, photopic negative response (PhNR), and sum of the oscillatory potentials (ΣOPs, sum of OP1-3 amplitudes) were analyzed. In addition, the implicit times of the a- and b-waves were also analyzed. RESULTS: The amplitudes and implicit times of all components of the fmERGs were significantly improved compared to the baseline at 3 months after beginning the intravitreal aflibercept injections (P < 0.0005-0.05). The amplitudes of the a-waves and PhNRs were further increased during the maintenance phase (P < 0.005-0.01). On the other hand, the amplitudes of the full-field a-waves and PhNR of the cone ERGs were significantly reduced at 6 and 12 months compared to the baseline. CONCLUSIONS: The macular function improved continuously during the maintenance phase of the intravitreal aflibercept injections. In contrast, the cone-driven functions of the more peripheral retina decreased with repeated injections suggesting adverse effects of the intravitreal aflibercept injections on the function of the more peripheral normal retina.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Macular Exsudativa/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Neovascularização de Coroide/diagnóstico por imagem , Neovascularização de Coroide/fisiopatologia , Eletrorretinografia , Feminino , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Tomografia de Coerência Óptica , Degeneração Macular Exsudativa/diagnóstico por imagem , Degeneração Macular Exsudativa/fisiopatologiaRESUMO
PURPOSE: To evaluate the physiology of the macular and whole retina after intravitreal aflibercept (IVAs) injections in patients with macular edema associated with a central retinal vein occlusion (CRVO) by electroretinography (ERG). METHODS: We studied 20 eyes of 20 patients with non-ischemic CRVO (72.0 ± 9.2 years). All patients were treated with monthly injections of IVA for the initial 3 months and then treated by the treat-and-extend (TAE) regimen for 12 months. The best-corrected visual acuity (BCVA), optical coherence tomographic images, focal macular ERGs (fmERGs), and full-field ERGs recorded before and after the treatment were compared. The fmERGs were elicited by a 15° white stimulus spot centered on the fovea. The full-field ERGs were recorded by a protocol recommended by International Society for Clinical Electrophysiology of Vision. The amplitudes and implicit times determined before and after the IVA were compared. RESULTS: The foveal thickness was significantly reduced accompanied by improvement of the BCVA after the treatments, and the improvements were maintained for at least 12 months. The amplitudes and implicit times of the fmERGs improved continuously for the 12 months. On the other hand, the reduced amplitudes of the full-field ERG, summed oscillatory potentials, and the photopic negative responses remained unchanged for the 12-month period. However, the implicit times of the maximum and cone responses were significantly shortened after the IVA. CONCLUSIONS: IVA injections by the TAE regimen led to a continuous improvement of the macular function in patients with ME associated with a CRVO. However, the function of the whole retina changed differently than the macula after the treatment.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Edema Macular/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/fisiopatologia , Oclusão da Veia Retiniana/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Eletrorretinografia , Feminino , Angiofluoresceinografia , Humanos , Injeções Intravítreas , Edema Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Oclusão da Veia Retiniana/fisiopatologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologiaRESUMO
Fish cell lines are widely used for the studies of developmental biology, virology, biology of aging, and nutrition physiology. However, little is known about their physicochemical properties. Here, we report the phospholipid compositions and mechanical properties of cell membranes derived from freshwater, anadromous and marine fish species. Biophysical analyses revealed that fish cell lines have highly deformable cell membranes with significantly low membrane tensions and Young's moduli compared with those of mammalian cell lines. The induction of cellular senescence by DNA demethylation using 5-Aza-2'-deoxycytidine significantly reduced the deformability of fish cell membrane, but hydrogen peroxide-induced oxidative stress did not affect the deformability. Mass spectrometry analysis of phospholipids revealed that the level of phosphatidylethanolamine molecules containing polyunsaturated fatty acids significantly increased during the 5-Aza-2'-deoxycytidine-induced cellular senescence. Fish cell lines provide a useful model system for studying the changes in the physicochemical properties of cell membranes during cellular senescence.Abbreviations: 2D-TLC: two-dimensional thin layer chromatography; 5-Aza-dC: 5-Aza-2'-deoxycytidine; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; FBS: fetal bovine serum; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PUFA: polyunsaturated fatty acid; SA-ß-gal: senescence-associated beta-galactosidase; SM: sphingomyelin.
Assuntos
Membrana Celular/metabolismo , Senescência Celular , Peixes , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Desmetilação do DNA , Decitabina/farmacologia , Ácidos Graxos/metabolismo , Lipídeos de Membrana/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismoRESUMO
In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins.
Assuntos
Diglicerídeos/metabolismo , Proteínas de Drosophila/metabolismo , Receptores de Lipoproteínas/metabolismo , Animais , Drosophila , Ácidos Graxos/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Lipoproteínas/metabolismo , Fosfolipídeos/metabolismoRESUMO
The Δ9-fatty acid desaturase introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how Δ9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole Δ9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca2+-dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of Δ9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.
Assuntos
Motivos de Aminoácidos/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Prolina/química , Proteólise , Animais , Regulação Enzimológica da Expressão Gênica , CamundongosRESUMO
The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia.
Assuntos
Canais de Cálcio/genética , Hipocalcemia/genética , Fibras Musculares Esqueléticas/patologia , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/patologia , Adulto , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Criança , Pré-Escolar , Células HEK293 , Heterozigoto , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Mutação de Sentido Incorreto , Miopatias Congênitas Estruturais/complicações , Proteína ORAI1 , Linhagem , Molécula 1 de Interação EstromalRESUMO
Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAß1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.
Assuntos
Distroglicanas/metabolismo , Glicosiltransferases/metabolismo , Laminina/metabolismo , Distrofias Musculares/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Animais , Sítios de Ligação , Encéfalo/enzimologia , Encéfalo/patologia , Células Cultivadas , Criança , Modelos Animais de Doenças , Distroglicanas/genética , Ensaios Enzimáticos , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Glicosiltransferases/genética , Humanos , Rim/enzimologia , Rim/patologia , Laminina/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Miocárdio/enzimologia , Miocárdio/patologia , N-Acetilglucosaminiltransferases/genética , Especificidade de Órgãos , Ligação ProteicaRESUMO
Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192âMet) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.
Assuntos
Distroglicanas/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação de Sentido Incorreto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Linhagem , Fenótipo , Análise de Sequência de DNARESUMO
α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.
Assuntos
Infecções por Arenaviridae/etiologia , Infecções por Arenaviridae/metabolismo , Distroglicanas/metabolismo , Laminina/metabolismo , Vírus da Coriomeningite Linfocítica , N-Acetilglucosaminiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Distroglicanas/química , Distroglicanas/genética , Glicosilação , Células HEK293 , Humanos , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Mioblastos/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/química , Internalização do VírusRESUMO
LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.
Assuntos
Distroglicanas/metabolismo , Glicosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Ácido Glucurônico/metabolismo , Glicosiltransferases/química , Concentração de Íons de Hidrogênio , Cinética , Camundongos , N-Acetilglucosaminiltransferases/química , Multimerização Proteica , Xilose/metabolismoRESUMO
Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures. We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca(2+) channel (Ca(v)2.3). EFHC1 and Ca(v)2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Ca(v)2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca(2+) currents that were reversed by the mutations associated with JME.
Assuntos
Epilepsia Mioclônica Juvenil/genética , Animais , Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Humanos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , LinhagemRESUMO
Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.
Assuntos
Canais Iônicos , Regeneração , Células Satélites de Músculo Esquelético , Animais , Camundongos , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Canais Iônicos/genética , Canais Iônicos/fisiologia , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Transdução de Sinais , Células Satélites de Músculo Esquelético/fisiologia , Regeneração/genética , Regeneração/fisiologiaRESUMO
Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.
Assuntos
Distroglicanas , Distrofias Musculares , N-Acetilglucosaminiltransferases , Animais , Camundongos , Distroglicanas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicosilação , Laminina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , N-Acetilglucosaminiltransferases/metabolismoRESUMO
PURPOSE: To determine whether there are significant correlations between the focal photopic negative response (PhNR), the focal visual sensitivity and the ganglion cell complex (GCC) thickness in glaucomatous eyes. STUDY DESIGN: Single-center observational study. METHODS: Fifty-two eyes of 52 patients (71.4 ± 9.42 years) with clinically diagnosed open angle glaucoma were studied. Thirty-six age-matched normal subjects served as controls. The focal PhNR of the focal macular electroretinograms (fmERGs) were elicited by a 15° circular, a superior semicircular or an inferior semicircular stimulus centered on the fovea. The thickness of the GCC was measured in the corresponding retinal areas in the spectral-domain optical coherence tomographic images. The visual sensitivities (dB) were measured by microperimetry at the retinal area where the fmERGs were elicited and were converted to liner values (1/Lambert). RESULTS: The focal PhNR amplitudes were significantly correlated with the visual sensitivities of the full-circle (R = 0.532), the superior (R = 0.530) and inferior (R = 0.526) semicircular responses (P < 0.0001). The GCC thickness was correlated with the visual sensitivities in the same areas with stronger correlations (R = 0.700, 0.759 and 0.650, respectively; P < 0.0001). The focal PhNR amplitudes were proportionally reduced with the thinning of the GCC thickness (R = 0.494, 0.518 and 0.511, respectively; P < 0.0001). CONCLUSIONS: The significant correlations between the focal PhNR amplitudes, the focal visual sensitivities and the GCC thickness indicate that these may be good biomarkers to track the changes in the physiology and anatomy of the macular area in glaucomatous eyes.
Assuntos
Glaucoma de Ângulo Aberto , Glaucoma , Eletrorretinografia , Glaucoma de Ângulo Aberto/diagnóstico , Humanos , Células Ganglionares da Retina , Tomografia de Coerência Óptica , Testes de Campo Visual , Campos VisuaisRESUMO
Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F1Fo-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F1Fo-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes.
Assuntos
Ácidos Graxos Dessaturases , Fosfolipídeos , Adenosina Trifosfatases , Animais , Drosophila , Estearoil-CoA Dessaturase , TemperaturaRESUMO
Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca2+ influx through Piezo1 and on the activation of the calcium-activated potassium channel KCa3.1. This suggests that Ca2+ ions can regulate EGF-stimulated macropinocytosis. We propose the potential for macropinocytosis inhibition through the regulation of a mechanosensitive channel activity using chemical tools.