RESUMO
GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.
Assuntos
Gangliosidose GM1 , Animais , Humanos , Camundongos , beta-Galactosidase/genética , beta-Galactosidase/metabolismo , beta-Galactosidase/uso terapêutico , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M1)/uso terapêutico , Gangliosidose GM1/genética , Glicolipídeos , Neuraminidase/genética , Neuraminidase/uso terapêuticoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.
Assuntos
COVID-19 , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Autofagia , Encéfalo/patologia , COVID-19/patologia , Células HeLa , Homeostase , Lisossomos , Doenças Neurodegenerativas/patologia , Fases de Leitura Aberta , SARS-CoV-2 , EsfingolipídeosRESUMO
The SUSD4 (Sushi domain-containing protein 4) gene encodes a complement inhibitor that is frequently deleted in 1q41q42 microdeletion syndrome, a multisystem congenital disorder that includes neurodevelopmental abnormalities. To understand SUSD4's role in the mammalian nervous system, we analyzed Susd4 knockout (KO) mice. Susd4 KO mice exhibited significant defects in motor performance and significantly higher levels of anxiety-like behaviors. Susd4 KO brain had abnormal "hairy" basket cells surrounding Purkinje neurons within the cerebellum and significantly reduced dendritic spine density in hippocampal pyramidal neurons. Neurons and oligodendrocyte lineage cells of wild-type mice were found to express Susd4 mRNA. Protein expression of the complement component C1q was increased in the brains of Susd4 KO mice. Our data indicate that SUSD4 plays an important role in neuronal functions, possibly via the complement pathway, and that SUSD4 deletion may contribute to the nervous system abnormalities in patients with 1q41q42 deletions.
RESUMO
Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Resistência à Doença/genética , Globosídeos/genética , Receptores de Hidrocarboneto Arílico/genética , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Triexosilceramidas/genética , Animais , Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Genoma Humano/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/biossíntese , Lipídeos/genética , Camundongos , Camundongos Knockout , Toxina Shiga/farmacologia , Transdução de Sinais/genética , Esfingolipídeos/genéticaRESUMO
Sphingolipids are membrane and bioactive lipids that are required for many aspects of normal mammalian development and physiology. However, the importance of the regulatory mechanisms that control sphingolipid levels in these processes is not well understood. The mammalian ORMDL proteins (ORMDL1, 2 and 3) mediate feedback inhibition of the de novo synthesis pathway of sphingolipids by inhibiting serine palmitoyl transferase in response to elevated ceramide levels. To understand the function of ORMDL proteins in vivo, we studied mouse knockouts (KOs) of the Ormdl genes. We found that Ormdl1 and Ormdl3 function redundantly to suppress the levels of bioactive sphingolipid metabolites during myelination of the sciatic nerve. Without proper ORMDL-mediated regulation of sphingolipid synthesis, severe dysmyelination results. Our data indicate that the Ormdls function to restrain sphingolipid metabolism in order to limit levels of dangerous metabolic intermediates that can interfere with essential physiological processes such as myelination.
Assuntos
Proteínas de Membrana/genética , Bainha de Mielina/genética , Esfingolipídeos/genética , Animais , Ceramidas/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipogênese/genética , Camundongos , Camundongos Knockout , Bainha de Mielina/metabolismo , Nervo Isquiático/crescimento & desenvolvimento , Nervo Isquiático/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Transdução de Sinais/genética , Esfingolipídeos/biossínteseRESUMO
Sphingolipids are a diverse class of essential cellular lipids that function as structural membrane components and as signaling molecules. Cells acquire sphingolipids by both de novo biosynthesis and recycling of exogenous sphingolipids. The individual importance of these pathways for the generation of essential sphingolipids in differentiated cells is not well understood. To investigate the requirement for de novo sphingolipid biosynthesis in adipocytes, a cell type with highly regulated lipid metabolism, we generated mice with an adipocyte-specific deletion of Sptlc1 Sptlc1 is an obligate subunit of serine palmitoyltransferase, the enzyme responsible for the first and rate-limiting step of de novo sphingolipid biosynthesis. These mice, which initially developed adipose tissue, exhibited a striking age-dependent loss of adipose tissue accompanied by evidence of adipocyte death, increased macrophage infiltration, and tissue fibrosis. Adipocyte differentiation was not affected by the Sptlc1 deletion. The mice also had elevated fasting blood glucose, fatty liver, and insulin resistance. Collectively, these data indicate that de novo sphingolipid biosynthesis is required for adipocyte cell viability and normal metabolic function and that reduced de novo sphingolipid biosynthesis within adipocytes is associated with adipocyte death, adipose tissue remodeling, and metabolic dysfunction.
Assuntos
Adipócitos/citologia , Homeostase , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Adiposidade , Animais , Diferenciação Celular , Sobrevivência Celular , Deleção de Genes , Inflamação , Resistência à Insulina , Metabolismo dos Lipídeos , Lipogênese , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Serina C-Palmitoiltransferase/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive ß-cell proliferation that was demonstrated after treatment with either a high-fat diet or the ß-cell-specific toxin, streptozotocin. Importantly, ß-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with ß-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes ß-cell endoplasmic reticulum stress, which is a known cause of ß-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes.
Assuntos
Proliferação de Células/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Dieta Hiperlipídica , Chaperona BiP do Retículo Endoplasmático , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Ilhotas Pancreáticas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoéster Fosfórico Hidrolases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Estreptozocina/farmacologiaRESUMO
Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic and neurodegenerative diseases, levels of some sphingolipids, including the bioactive metabolite ceramide, are elevated. Sphingolipid metabolism has been linked to autophagy, a critical catabolic process in both normal cell function and disease; however, the in vivo relevance of the interaction is not well-understood. Here, we show that blocking autophagy in the liver by deletion of the Atg7 gene, which is essential for autophagosome formation, causes an increase in sphingolipid metabolites including ceramide. We also show that overexpression of serine palmitoyltransferase to elevate de novo sphingolipid biosynthesis induces autophagy in the liver. The results reveal autophagy as a process that limits excessive ceramide levels and that is induced by excessive elevation of de novo sphingolipid synthesis in the liver. Dysfunctional autophagy may be an underlying mechanism causing elevations in ceramide that may contribute to pathogenesis in diseases.
Assuntos
Autofagia , Fígado/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/metabolismo , Animais , Proteína 7 Relacionada à Autofagia , Ceramidas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fígado/enzimologia , Fígado/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Microssomos Hepáticos/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Mutantes/metabolismo , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Serina C-Palmitoiltransferase/genéticaRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphorylated by the actions of two S1P-specific phosphatases, sphingosine-1-phosphate phosphatases 1 and 2. To identify the physiological functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1(-/-) mice appeared normal at birth, but during the 1st week of life they exhibited stunted growth and suffered desquamation, with most dying before weaning. Both Sgpp1(-/-) pups and surviving adults exhibited multiple epidermal abnormalities. Interestingly, the epidermal permeability barrier developed normally during embryogenesis in Sgpp1(-/-) mice. Keratinocytes isolated from the skin of Sgpp1(-/-) pups had increased intracellular S1P levels and displayed a gene expression profile that indicated overexpression of genes associated with keratinocyte differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis.
Assuntos
Diferenciação Celular/genética , Epiderme/metabolismo , Queratinócitos/metabolismo , Proteínas de Membrana/genética , Monoéster Fosfórico Hidrolases/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Análise por Conglomerados , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Queratinócitos/citologia , Lisofosfolipídeos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Monoéster Fosfórico Hidrolases/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/embriologia , Pele/crescimento & desenvolvimento , Pele/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de TempoRESUMO
Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.
Assuntos
Aldeído Liases , Movimento Celular/imunologia , Lisofosfolipídeos/metabolismo , Neutrófilos , Transdução de Sinais/imunologia , Esfingosina/análogos & derivados , Proteínas de Fase Aguda/imunologia , Aldeído Liases/genética , Aldeído Liases/imunologia , Aldeído Liases/metabolismo , Animais , Biomarcadores/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Hematopoese/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Lisofosfolipídeos/imunologia , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Esfingosina/imunologia , Esfingosina/metabolismoRESUMO
Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.
Assuntos
Anafilaxia/enzimologia , Regulação Enzimológica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Transplante de Medula Óssea , Taxa de Filtração Glomerular , Células-Tronco Hematopoéticas/metabolismo , Histamina/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , PermeabilidadeRESUMO
S1P1 receptor expression is required for the egress of newly formed T cells from the thymus and exit of mature T and B cells from secondary lymphoid organs. In this study, we deleted the expression of the S1P1 receptor gene (S1pr1) in developing B cells in the bone marrow. Although B cell maturation within the bone marrow was largely normal in the B cell-specific S1pr1 knockout (B-S1pr1KO) mice, their newly generated immature B cells appeared in the blood at abnormally low numbers as compared with control mice. In the bone marrow of B-S1pr1KO mice, immature B cells in contact with the vascular compartment displayed increased apoptosis as compared with control mice. Forced expression of CD69, a negative regulator of S1P1 receptor expression, in developing bone marrow B cells also reduced the number of immature B cells in the blood. Attenuation of CXCR4 signaling, which is required for the proper retention of developing B cells in bone marrow, did not release immature B cells into the blood of B-S1pr1KO mice as effectively as in control mice. Our results indicate that the S1P1 receptor provides a signal necessary for the efficient transfer of newly generated immature B cells from the bone marrow to the blood.
Assuntos
Medula Óssea/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Lisoesfingolipídeo/genética , Animais , Apoptose , Deleção de Genes , Regulação da Expressão Gênica , Homeostase , Integrases/genética , Camundongos , Camundongos Knockout , Células Precursoras de Linfócitos B/fisiologia , Receptores de Lisoesfingolipídeo/deficiência , Receptores de Lisoesfingolipídeo/fisiologia , Receptores de Esfingosina-1-Fosfato , Baço/imunologia , Linfócitos T/imunologia , Timo/imunologiaRESUMO
Molecular modeling and site directed mutagenesis were used to analyze the structural features determining the unique inhibitor sensitivities of type-III phosphatidylinositol 4-kinase enzymes (PI4Ks). Mutation of a highly conserved Tyr residue that provides the bottom of the hydrophobic pocket for ATP yielded a PI4KIIIbeta enzyme that showed greatly reduced wortmannin sensitivity and was catalytically still active. Similar substitutions were not tolerated in the type-IIIalpha enzyme rendering it catalytically inactive. Two conserved Cys residues located in the active site of PI4KIIIalpha were found responsible for the high sensitivity of this enzyme to the oxidizing agent, phenylarsine oxide. Mutation of one of these Cys residues reduced the phenylarsine oxide sensitivity of the enzyme to the same level observed with the PI4KIIIbeta protein. In search of inhibitors that would discriminate between the closely related PI4KIIIalpha and -IIIbeta enzymes, the PI3Kgamma inhibitor, PIK93, was found to inhibit PI4KIIIbeta with significantly greater potency than PI4KIIIalpha. These studies should aid development of subtype-specific inhibitors of type-III PI4Ks and help to better understand the significance of localized PtdIns4P production by the various PI4Ks isoforms in specific cellular compartments.
Assuntos
1-Fosfatidilinositol 4-Quinase/genética , Alelos , Modelos Moleculares , 1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/química , Sequência de Aminoácidos , Androstadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Homologia de Sequência de Aminoácidos , WortmaninaRESUMO
The S1P1 receptor, on the surface of lymphocytes and endothelial cells, regulates the unique trafficking behavior of certain lymphocyte populations. We have examined whether the S1P1 receptor also dictates the distinctive tissue distribution of V alpha14-J alpha18 natural killer T (NKT) cells, whose trafficking pattern is not well understood. Mice (TCS1P1 KO) were established with a conditional deletion of the S1P1 receptor in thymocytes that included precursors of NKT cells. Within the thymus, NKT cells were found at normal or increased levels, indicating that S1P1 receptor expression was dispensable for NKT cell development. However, substantially reduced numbers of NKT cells were detected in the peripheral tissues of the TCS1P1 KO mice. Short-term S1P1 deletion after NKT cells had established residence in the periphery did not substantially alter their distribution in tissues, except for a partial decrease in the spleen. FTY720, a S1P1 receptor ligand that has potent effects on the trafficking of conventional T cells, did not alter the preexisting distribution of NKT cells within peripheral tissues of wild-type mice. Our results indicate that the S1P1 receptor expression on NKT cells is dispensable for development within thymus but is essential for the establishment of their tissue residency in the periphery.
Assuntos
Células Matadoras Naturais/citologia , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/fisiologia , Transdução de SinaisRESUMO
The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.
Assuntos
1-Fosfatidilinositol 4-Quinase/química , Proteínas de Transporte/química , Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Receptores de Esteroides/química , 1-Fosfatidilinositol 4-Quinase/metabolismo , Acetilcisteína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Androstadienos/farmacologia , Animais , Western Blotting , Células COS , Cálcio/química , Cálcio/metabolismo , Cálcio/farmacologia , Quelantes/farmacologia , Chlorocebus aethiops , Citoplasma/metabolismo , DNA/metabolismo , Regulação para Baixo , Ácido Egtázico/farmacologia , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Ionomicina/farmacologia , Ionóforos , Metabolismo dos Lipídeos , Microscopia Confocal , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Frações Subcelulares , Fatores de Tempo , Transfecção , WortmaninaRESUMO
The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.
Assuntos
Membrana Celular/metabolismo , Lipídeos/química , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Células COS , Cálcio/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Transferência de Energia , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde , Humanos , Concentração Inibidora 50 , Inositol/metabolismo , Isoenzimas/metabolismo , Metabolismo dos Lipídeos , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C delta , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Espectrometria de Fluorescência , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Phosphorylation of phosphatidylinositol (PI) to PI 4-phosphate is one of the key reactions in the production of phosphoinositides, lipid regulators of several cellular functions. This reaction is catalyzed by multiple enzymes that belong either to the type II or the type III family of PI 4-kinases. Type III enzymes are structurally similar to PI 3-kinases and are sensitive to PI 3-kinase inhibitors. In contrast, the recent cloning of the first type II PI 4-kinase enzyme defined a novel enzyme family. Here we characterize a new member of this family, the type IIbeta enzyme that has been identified in the NCBI data base based on its homology to the first-cloned type IIalpha enzyme. The type IIbeta enzyme has a primary transcript size of approximately 3.8 kb and shows wide tissue distribution. It contains an open reading frame of 1.4 kb, encoding a protein of approximately 54 kDa. Sequence comparison reveals a high degree of similarity to the type IIalpha enzyme within the C-terminal catalytic domain but significantly lower homology within the N-terminal region. Expression of both enzyme yields increased PI 4-kinase activity that is associated with the microsomal membrane fractions and is significantly lower for the type IIbeta than the type IIalpha form. Both enzymes use PI as their primary substrate and have no detectable activity on PI monophosphates. Epitope-tagged as well as green fluorescent protein-tagged forms of both enzymes localize primarily to intracellular membranes and show prominent co-localization with early endosomes and recycling endosomes but not with the Golgi. These compartments participate in the processing of both the transferrin receptor and the G protein-coupled AT(1A) angiotensin receptor. Our data indicate the existence of multiple forms of type II PI 4-kinase in mammalian cells and suggest that their functions are related to the endocytic pathway.