RESUMO
The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , MicroRNAs/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Proteínas Argonautas , Dendritos/metabolismo , Proteína 4 Homóloga a Disks-Large , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/fisiologia , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Guanilato Quinases , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Fosforilação , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Resposta ao Choque Térmico , Fatores de Terminação de Peptídeos/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Fatores de Terminação de Peptídeos/genética , Príons/genética , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autopsia , Western Blotting , Encéfalo/patologia , Encéfalo/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Sarcosina/análogos & derivados , Sarcosina/química , SolubilidadeRESUMO
A- and C-type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C-type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A-type lamin channels interact with several nuclear components, including fibrillarin- and UBF-positive regions of nucleoli, foci of heterochromatin protein 1 ß, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C-type lamin channels and nuclear pores, lamin-associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C-type lamins had low levels of B-type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell-specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures.
Assuntos
Núcleo Celular/genética , Reparo do DNA/genética , Lamina Tipo A/ultraestrutura , Lamina Tipo B/ultraestrutura , Animais , Proteínas Cromossômicas não Histona/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , CamundongosRESUMO
Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPß and the lack of known ligands for the latter, we examined Sp-D binding to SIRPß. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPß. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Cálcio/metabolismo , Neutrófilos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores Imunológicos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/genética , Antígeno CD47/genética , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Células HL-60 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteína D Associada a Surfactante Pulmonar/genética , Receptores Imunológicos/genética , Deleção de SequênciaRESUMO
The adenylyl cyclase stimulator forskolin (FSK) stimulates UT-A1 phosphorylation, membrane trafficking, and urea transport activity. Here, we found that FSK stimulation induces UT-A1 ubiquitination in UT-A1 Madin-Darby canine kidney (MDCK) cells. This suggests that phosphorylation by FSK also triggers the protein degradation machinery for UT-A1. UT-A1-MDCK cells were treated with 100 µg/ml cycloheximide to inhibit protein synthesis, with or without 10 µM FSK. Total UT-A1 protein abundance was significantly reduced after FSK treatment, concomitantly ubiquitinated UT-A1 was increased. We then specifically investigated the effect of FSK on UT-A1 expressed on the cell plasma membrane. FSK treatment accelerated UT-A1 removal from the cell plasma membrane by increasing UT-A1 endocytosis as judged by biotinylation/MesNa treatment and confocal microscopy. We further found that inhibition of the clathrin-mediated endocytic pathway, but not the caveolin-mediated endocytic pathway, significantly blocks FSK-stimulated UT-A1 endocytosis. The PKA inhibitor H89 and the proteasome inhibitors MG132 and lactacystin reduced FSK-induced membrane UT-A1 reduction. Our study shows that FSK activates the UT-A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation of UT-A1, which represents an important mechanism for the cell to return to the basal conditions after vasopressin stimulation.
Assuntos
Colforsina/farmacologia , Endocitose/efeitos dos fármacos , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteólise/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Clatrina/farmacologia , Cicloeximida/farmacologia , Cães , Rim/citologia , Rim/efeitos dos fármacos , Modelos Animais , Transdução de Sinais/efeitos dos fármacos , Transportadores de UreiaRESUMO
Interaction of SIRPα with its ligand, CD47, regulates leukocyte functions, including transmigration, phagocytosis, oxidative burst, and cytokine secretion. Recent progress has provided significant insights into the structural details of the distal IgV domain (D1) of SIRPα. However, the structural roles of proximal IgC domains (D2 and D3) have been largely unstudied. The high degree of conservation of D2 and D3 among members of the SIRP family as well as the propensity of known IgC domains to assemble in cis has led others to hypothesize that SIRPα forms higher order structures on the cell surface. Here we report that SIRPα forms noncovalently linked cis homodimers. Treatment of SIRPα-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRPα dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRPα, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRPα molecules revealed that SIRPα forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRPα being expressed on the cell surface as a functional cis-linked dimer.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Neutrófilos/metabolismo , Multimerização Proteica/fisiologia , Receptores Imunológicos/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígeno CD47/genética , Células CHO , Adesão Celular/fisiologia , Cricetinae , Cricetulus , Células HEK293 , Células HL-60 , Humanos , Mutação , Ativação de Neutrófilo/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC-IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable lentiviral vector-transduced derivatives of HEK-293T cells. The expression design includes allele-specific peptide ligands tethered to the amino-terminus of the MHC-II ß chain via a protease-cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide exchange, enabling high-throughput production of many distinct MHC-IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label-free methods, native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp multimer reagents. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and expression line creation Support Protocol 1: Six-well assay for estimation of production cell line yield Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His-tags Basic Protocol 2: Cultures for production of Class II MHC proteins Basic Protocol 3: Purification of Class II MHC proteins by anti-leucine zipper affinity chromatography Alternate Protocol 1: IMAC purification of His-tagged Class II MHC Support Protocol 3: Protein concentration measurements and adjustments Support Protocol 4: Polishing purification by anion-exchange chromatography Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation Basic Protocol 4: Peptide exchange Basic Protocol 5: Analysis of peptide exchange by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Alternate Protocol 2: Native isoelectric focusing to validate MHC-II peptide loading Basic Protocol 6: Multimerization Basic Protocol 7: Staining cells with Class II MHC tetramers.
Assuntos
Escherichia coli , Antígenos de Histocompatibilidade Classe II , Células HEK293 , Humanos , Indicadores e Reagentes , Coloração e RotulagemRESUMO
Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that approximately 50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.
Assuntos
Linfócitos B/imunologia , Regulação para Baixo , Antígenos HLA-D/biossíntese , Adulto , Linfócitos B/citologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Diferenciação Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Centro Germinativo/imunologia , Antígenos HLA-D/imunologia , Humanos , Monócitos/citologia , Monócitos/imunologiaRESUMO
BACKGROUND: Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to mu-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood. RESULTS: We recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Galphai/o complex, MOR1K couples to the stimulatory Galphas complex. CONCLUSION: The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.
Assuntos
Processamento Alternativo , Analgésicos Opioides/farmacologia , Morfina/farmacologia , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/genética , Analgésicos Opioides/metabolismo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Humanos , Morfina/metabolismo , Óxido Nítrico/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Opioides mu/agonistasRESUMO
Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.
Assuntos
Apoptose , Colo/citologia , Colo/metabolismo , Desmogleína 2/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Linhagem Celular , Desmogleína 2/química , Desmogleína 2/genética , Desmogleína 2/imunologia , Regulação para Baixo , Epitélio/metabolismo , Humanos , Junções Intercelulares/metabolismo , Microdomínios da Membrana/metabolismo , Dados de Sequência Molecular , RNA Interferente Pequeno/genética , Regulação para Cima , gama Catenina/metabolismoRESUMO
Neuroblastoma is an aggressive pediatric malignancy of the neural crest with suboptimal cure rates and a striking predilection for widespread metastases, underscoring the need to identify novel therapeutic vulnerabilities. We recently identified the RNA binding protein LIN28B as a driver in high-risk neuroblastoma and demonstrated it promotes oncogenic cell proliferation by coordinating a RAN-Aurora kinase A network. Here, we demonstrate that LIN28B influences another key hallmark of cancer, metastatic dissemination. Using a murine xenograft model of neuroblastoma dissemination, we show that LIN28B promotes metastasis. We demonstrate that this is in part due to the effects of LIN28B on self-renewal and migration, providing an understanding of how LIN28B shapes the metastatic phenotype. Our studies reveal that the let-7 family, which LIN28B inhibits, decreases self-renewal and migration. Next, we identify PDZ Binding Kinase (PBK) as a novel LIN28B target. PBK is a serine/threonine kinase that promotes the proliferation and self-renewal of neural stem cells and serves as an oncogenic driver in multiple aggressive malignancies. We demonstrate that PBK is both a novel direct target of let-7i and that MYCN regulates PBK expression, thus elucidating two oncogenic drivers that converge on PBK. Functionally, PBK promotes self-renewal and migration, phenocopying LIN28B. Taken together, our findings define a role for LIN28B in neuroblastoma metastasis and define the targetable kinase PBK as a potential novel vulnerability in metastatic neuroblastoma.
RESUMO
Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.
Assuntos
Proteínas de Transporte/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Citoesqueleto , Meiose , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Temperatura , UbiquitinaçãoRESUMO
Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and plays an important role in the maintenance of body water homeostasis. Excessive glucocorticoid as often seen in Cushing's syndrome causes water retention. However, whether and how glucocorticoid regulates AQP2 remains unclear. In this study, we examined the direct effect of dexamethasone on AQP2 protein expression and activity. Dexamethasone increased AQP2 protein abundance in rat inner medullary collecting duct (IMCD) suspensions. This was confirmed in HEK293 cells transfected with AQP2 cDNA. Cell surface protein biotinylation showed an increase of dexamethasone-induced cell membrane AQP2 expression and this effect was blocked by glucocorticoid receptor antagonist RU486. Functionally, dexamethasone treatment of oocytes injected with an AQP2 cRNA increased water transport activity as judged by cell rupture time in a hypo-osmotic solution (66 ± 13 s in dexamethasone vs. 101 ± 11 s in control, n = 15). We further found that dexamethasone treatment reduced AQP2 protein degradation, which could result in an increase of AQP2 protein. Interestingly, dexamethasone promoted cell membrane AQP2 moving to less buoyant lipid raft submicrodomains. Taken together, our data demonstrate that dexamethasone promotes AQP2 protein expression and increases water permeability mainly via inhibition of AQP2 protein degradation. The increase in AQP2 activity promotes water reabsorption, which may contribute to glucocorticoid-induced water retention and hypertension.
RESUMO
Junctional adhesion molecule-A (JAM-A) is a tight junction-associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.
Assuntos
Moléculas de Adesão Celular/metabolismo , Multimerização Proteica/fisiologia , Receptores de Superfície Celular/metabolismo , Junções Íntimas/fisiologia , Proteínas rap de Ligação ao GTP/biossíntese , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Células CHO , Adesão Celular/fisiologia , Moléculas de Adesão Celular/genética , Agregação Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular , Cricetulus , Células HEK293 , Humanos , Junções Intercelulares/metabolismo , Microscopia de Força Atômica , Mutação , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Transdução de Sinais , Junções Íntimas/genéticaRESUMO
The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. ß-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/ß-catenin signaling. Here we show that inhibition of Akt/ß-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated ß-catenin 552 (pß-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits ß-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes ß-catenin transactivation through Akt-dependent C-terminal phosphorylation of ß-catenin to promote its association with 14.3.3ζ. Augmented ß-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/ß-catenin from the nucleus, thereby inhibiting ß-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.
Assuntos
Proteínas 14-3-3/metabolismo , Interferon gama/farmacologia , Mucosa Intestinal/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/antagonistas & inibidores , Animais , Células CHO , Linhagem Celular , Proliferação de Células , Cricetulus , Ativação Enzimática , Inflamação , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de SinaisRESUMO
Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.
Assuntos
Vetores Genéticos/imunologia , Epitopos Imunodominantes/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Antígenos Virais/imunologia , Brefeldina A/imunologia , Células Cultivadas , Cricetinae , Citomegalovirus/imunologia , Replicação do DNA/imunologia , Células Dendríticas/imunologia , Vetores Genéticos/genética , Humanos , Interferon gama/imunologia , Ativação Linfocitária/imunologia , Monócitos/imunologia , Orthomyxoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral , Vírus da Febre Amarela/imunologiaRESUMO
The desmosomal cadherin desmoglein-2 (Dsg2) is a transmembrane cell adhesion protein that is widely expressed in epithelial and non-epithelial tissues, such as the intestine, epidermis, testis, and heart. Dsg2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis, and we have previously reported that intracellular fragments of Dsg2 promote apoptosis in colonic epithelial cells. While several studies have shown that both the extracellular and intracellular domains of Dsg2 can be targeted by proteases, identification of these putative Dsg2 fragments in colonic epithelial cells has not been performed. Here, we report that the mouse monoclonal antibody (mAb) AH12.2 binds to the first extracellular domain of Dsg2. Using this antibody along with previously described mAb against the extracellular (6D8) and intracellular (DG3.10) domains of Dsg2, we characterize the expression and identify the cleavage fragments of Dsg2 in colonic epithelial cells. This study provides a detailed description of the extracellular and intracellular Dsg2 cleavage fragments that are generated in the simple epithelium of the colon and will guide future studies examining the relationship of these fragments to cellular fate and disease states.
Assuntos
Colo/metabolismo , Desmogleína 2/metabolismo , Epitélio/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Anticorpos Monoclonais Murinos/metabolismo , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Células CHO , Linhagem Celular Transformada , Clonagem Molecular , Cricetinae , Desmossomos/metabolismo , Humanos , Immunoblotting , Camundongos , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Coordinated regulation of cell proliferation is vital for epithelial tissue homeostasis, and uncontrolled proliferation is a hallmark of carcinogenesis. A growing body of evidence indicates that epithelial tight junctions (TJs) play a role in these processes, although the mechanisms involved are poorly understood. In this study, we identify and characterize a novel plasma membrane pool of cyclin D1 with cell-cycle regulatory functions. We have determined that the zonula occludens (ZO) family of TJ plaque proteins sequesters cyclin D1 at TJs during mitosis, through an evolutionarily conserved class II PSD-95, Dlg, and ZO-1 (PDZ)-binding motif within cyclin D1. Disruption of the cyclin D1/ZO complex through mutagenesis or siRNA-mediated suppression of ZO-3 resulted in increased cyclin D1 proteolysis and G(0)/G(1) cell-cycle retention. This study highlights an important new role for ZO family TJ proteins in regulating epithelial cell proliferation through stabilization of cyclin D1 during mitosis.
Assuntos
Proteínas de Transporte/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Sequência de Aminoácidos , Animais , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Colo/citologia , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitose , Domínios PDZ , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas da Zônula de OclusãoRESUMO
The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of functionally distinct APC populations exist in the mucosal lamina propria (LP) below the intestinal epithelium, but their relative contributions to inflammation remain unclear. Here, we demonstrate in mice important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was found to be expressed in resident LP macrophages (defined as CD11b(+)F4/80(+)) but not DCs (defined as CD11c(+)CD103(+)). LP macrophage frequency and number were decreased in two strains of CX3CR1-knockout mice and in mice deficient in the CX3CR1 ligand CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS-induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL-17A antibodies or transfer of CX3CR1-sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders such as IBD.