RESUMO
Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death.
Assuntos
Morte Celular , Canais de Cloreto , Dependovirus , Células Ganglionares da Retina , Animais , Células Ganglionares da Retina/metabolismo , Dependovirus/genética , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Morte Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Masculino , RNA Interferente Pequeno/genéticaRESUMO
In pancreatic ß cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in ß cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gß5, such that the knockout of the Gnb5 gene results in degradation of all R7 subunits. We found that Gnb5 knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gß5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in Gnb5-/- MIN6 cells showed that cAMP, diacylglycerol, or Ca2+ levels were not significantly affected. There was no reduction in the amplitude of free Ca2+ responses in islets from the Gnb5-/- mice, but the frequency of Ca2+ oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of Gnb5 impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in Gnb5-/- cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gß5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic ß-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gß5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.
Assuntos
Sinalização do Cálcio/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas RGS/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , AMP Cíclico/genética , AMP Cíclico/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologia , Proteínas RGS/genética , Receptor Muscarínico M3/genéticaRESUMO
Tescalcin (TESC, also known as calcineurin-homologous protein 3, CHP3) is a 24-kDa EF-hand Ca2+-binding protein that has recently emerged as a regulator of cell differentiation and growth. The TESC gene has also been linked to human brain abnormalities, and high expression of tescalcin has been found in several cancers. The expression level of tescalcin changes dramatically during development and upon signal-induced cell differentiation. Recent studies have shown that tescalcin is not only subjected to up- or down-regulation, but also has an active role in pathways that drive cell growth and differentiation programs. At the molecular level, there is compelling experimental evidence showing that tescalcin can directly interact with and regulate the activities of the Na+/H+ exchanger NHE1, subunit 4 of the COP9 signalosome (CSN4) and protein kinase glycogen-synthase kinase 3 (GSK3). In hematopoetic precursor cells, tescalcin has been shown to couple activation of the extracellular signal-regulated kinase (ERK) cascade to the expression of transcription factors that control cell differentiation. The purpose of this Commentary is to summarize recent efforts that have served to characterize the biochemical, genetic and physiological attributes of tescalcin, and its unique role in the regulation of various cellular functions.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Motivos EF Hand , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/química , Diferenciação Celular/genética , Proliferação de Células , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/metabolismo , HumanosRESUMO
The Ca(2+)-binding protein tescalcin is known to be involved in hematopoietic cell differentiation; however, this mechanism is poorly understood. Here, we identify CSN4 (subunit 4 of the COP9 signalosome) as a novel binding partner of tescalcin. The COP9 signalosome (CSN) is a multiprotein complex that is essential for development in all eukaryotes. This interaction is selective, Ca(2+)-dependent and involves the PCI domain of CSN4 subunit. We then investigated tescalcin and CSN activity in human erythroleukemia HEL and promyelocytic leukemia K562 cells and find that phorbol 12-myristate 13-acetate (PMA)-induced differentiation, resulting in the upregulation of tescalcin, coincides with reduced deneddylation of cullin-1 (Cul1) and stabilization of p27(Kip1) - molecular events that are associated with CSN activity. The knockdown of tescalcin led to an increase in Cul1 deneddylation, expression of F-box protein Skp2 and the transcription factor c-Jun, whereas the levels of cell cycle regulators p27(Kip1) and p53 decreased. These effects are consistent with the hypothesis that tescalcin might play a role as a negative regulator of CSN activity towards Cul1 in the process of induced cell differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Culina/metabolismo , Hematopoese , Complexos Multiproteicos/metabolismo , Complexo do Signalossomo COP9 , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica/genética , Hematopoese/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células K562 , Ligação Proteica/genética , RNA Interferente Pequeno/genética , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We investigated the physiological role of Gß5, a unique G protein ß subunit that dimerizes with regulators of G protein signaling (RGS) proteins of the R7 family instead of Gγ. Gß5 is essential for stability of these complexes, so that its knockout (KO)causes degradation of the entire Gß5-R7 family. We report that the Gß5-KO mice remain leaner than the wild type (WT) throughout their lifetime and are resistant to a high-fat diet. They have a 5-fold increase in locomotor activity, increased thermogenesis, and lower serum insulin, all of which correlate with a higher level of secreted epinephrine. Heterozygous (HET) mice are 2-fold more active than WT mice. Surprisingly, with respect to body weight, the HET mice display a phenotype opposite to that of the KO mice: by the age of 6 mo, they are ≥ 15% heavier than the WT and have increased adiposity, insulin resistance, and liver steatosis. These changes occur in HET mice fed a normal diet and without apparent hyperphagia, mimicking basic characteristics of human metabolic syndrome. We conclude that even a partial reduction in Gß5-R7 level can perturb normal animal metabolism and behavior. Our data on Gß5 haploinsufficient mice may explain earlier observations of genetic linkage between R7 family mutations and obesity in humans.
Assuntos
Comportamento Animal , Peso Corporal/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Atividade Motora , Animais , Glicemia/metabolismo , Catecolaminas/urina , Dieta Hiperlipídica , Ingestão de Alimentos , Metabolismo Energético , Epinefrina/metabolismo , Heterozigoto , Insulina/sangue , Camundongos , Camundongos KnockoutRESUMO
Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular/genética , Granulócitos/fisiologia , Células HL-60/fisiologia , Macrófagos/fisiologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Humanos , Macrófagos/metabolismo , RNA Interferente Pequeno/farmacologia , Tretinoína/farmacologia , Células U937 , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation, such as expression of specific cell surface antigens, inhibition of cell proliferation, and polyploidization. Conversely, knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin, the expression of Fli-1, Ets-1, and Ets-2 transcription factors, but not GATA-1 or MafB, is blocked. Thus, tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Megacariócitos/citologia , Megacariócitos/metabolismo , Telomerase/classificação , Telomerase/genética , Antígenos CD34/metabolismo , Células da Medula Óssea/metabolismo , Proteínas de Ligação ao Cálcio/genética , Adesão Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Integrinas/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Poliploidia , Telomerase/metabolismo , Transcrição Gênica/genéticaRESUMO
Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).
Assuntos
Cálcio/química , Receptor Quinase 1 Acoplada a Proteína G/fisiologia , Recoverina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Receptor Quinase 1 Acoplada a Proteína G/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Ligação Proteica , Rodopsina/químicaRESUMO
The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca(2+) ion.
Assuntos
Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/química , Cálcio/química , Motivos EF Hand , Magnésio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Inibidores de Calcineurina , Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Cátions Bivalentes/química , Dicroísmo Circular , Cricetinae , Diálise , Motivos EF Hand/genética , Ativação Enzimática , Células HL-60 , Células HeLa , Humanos , Soros Imunes/biossíntese , Soros Imunes/isolamento & purificação , Células K562 , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Células PC12 , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de Proteína , Espectrometria de Fluorescência , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Triptofano/químicaRESUMO
RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.