RESUMEN
Cells subjected to environmental stresses undergo regulated cell death (RCD) when homeostatic programs fail to maintain viability. A major mechanism of RCD is the excessive calcium loading of mitochondria and consequent triggering of the mitochondrial permeability transition (mPT), which is especially important in post-mitotic cells such as cardiomyocytes and neurons. Here, we show that stress-induced upregulation of the ROS-generating protein Nox4 at the ER-mitochondria contact sites (MAMs) is a pro-survival mechanism that inhibits calcium transfer through InsP3 receptors (InsP3 R). Nox4 mediates redox signaling at the MAM of stressed cells to augment Akt-dependent phosphorylation of InsP3 R, thereby inhibiting calcium flux and mPT-dependent necrosis. In hearts subjected to ischemia-reperfusion, Nox4 limits infarct size through this mechanism. These results uncover a hitherto unrecognized stress pathway, whereby a ROS-generating protein mediates pro-survival effects through spatially confined signaling at the MAM to regulate ER to mitochondria calcium flux and triggering of the mPT.
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Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , NADPH Oxidasa 4/metabolismo , Animales , Supervivencia Celular , Receptores de Inositol 1,4,5-Trifosfato/genética , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , NADPH Oxidasa 4/genética , Estrés Oxidativo , RatasRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1008328.].
RESUMEN
Candida albicans cells depend on the energy derived from amino acid catabolism to induce and sustain hyphal growth inside phagosomes of engulfing macrophages. The concomitant deamination of amino acids is thought to neutralize the acidic microenvironment of phagosomes, a presumed requisite for survival and initiation of hyphal growth. Here, in contrast to an existing model, we show that mitochondrial-localized NAD+-dependent glutamate dehydrogenase (GDH2) catalyzing the deamination of glutamate to α-ketoglutarate, and not the cytosolic urea amidolyase (DUR1,2), accounts for the observed alkalization of media when amino acids are the sole sources of carbon and nitrogen. C. albicans strains lacking GDH2 (gdh2-/-) are viable and do not extrude ammonia on amino acid-based media. Environmental alkalization does not occur under conditions of high glucose (2%), a finding attributable to glucose-repression of GDH2 expression and mitochondrial function. Consistently, inhibition of oxidative phosphorylation or mitochondrial translation by antimycin A or chloramphenicol, respectively, prevents alkalization. GDH2 expression and mitochondrial function are derepressed as glucose levels are lowered from 2% (~110 mM) to 0.2% (~11 mM), or when glycerol is used as primary carbon source. Using time-lapse microscopy, we document that gdh2-/- cells survive, filament and escape from primary murine macrophages at rates indistinguishable from wildtype. In intact hosts, such as in fly and murine models of systemic candidiasis, gdh2-/- mutants are as virulent as wildtype. Thus, although Gdh2 has a critical role in central nitrogen metabolism, Gdh2-catalyzed deamination of glutamate is surprisingly dispensable for escape from macrophages and virulence. Consistently, using the pH-sensitive dye (pHrodo), we observed no significant difference between wildtype and gdh2-/- mutants in phagosomal pH modulation. Following engulfment of fungal cells, the phagosomal compartment is rapidly acidified and hyphal growth initiates and sustained under consistently acidic conditions within phagosomes. Together, our results demonstrate that amino acid-dependent alkalization is not essential for hyphal growth, survival in macrophages and hosts. An accurate understanding of the microenvironment within macrophage phagosomes and the metabolic events underlying the survival of phagocytized C. albicans cells and their escape are critical to understanding the host-pathogen interactions that ultimately determine the pathogenic outcome.
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Candida albicans/inmunología , Candidiasis/inmunología , Drosophila melanogaster/inmunología , Glutamato Deshidrogenasa/metabolismo , Macrófagos/inmunología , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Candida albicans/patogenicidad , Candidiasis/metabolismo , Candidiasis/microbiología , Drosophila melanogaster/metabolismo , Drosophila melanogaster/microbiología , Femenino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutamato Deshidrogenasa/genética , Interacciones Huésped-Patógeno , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Nitrógeno , Fagosomas/inmunología , Fagosomas/metabolismo , Fagosomas/microbiología , VirulenciaRESUMEN
Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine-threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species-generating NADPH oxidase-4 (Nox4) is induced downstream of ATF4, binds to a PP1-targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4-regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia-reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4-GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
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Factor 2 Eucariótico de Iniciación/metabolismo , NADPH Oxidasas/metabolismo , Proteína Fosfatasa 1/metabolismo , Receptores de Neuropéptido Y/antagonistas & inhibidores , Transducción de Señal , Animales , Línea Celular , Humanos , NADPH Oxidasa 4 , Oxidación-ReducciónRESUMEN
BACKGROUND: Extracellular matrix (ECM) remodeling contributes to in-stent restenosis and thrombosis. Despite its important clinical implications, little is known about ECM changes post-stent implantation. METHODS: Bare-metal and drug-eluting stents were implanted in pig coronary arteries with an overstretch under optical coherence tomography guidance. Stented segments were harvested 1, 3, 7, 14, and 28 days post-stenting for proteomics analysis of the media and neointima. RESULTS: A total of 151 ECM and ECM-associated proteins were identified by mass spectrometry. After stent implantation, proteins involved in regulating calcification were upregulated in the neointima of drug-eluting stents. The earliest changes in the media were proteins involved in inflammation and thrombosis, followed by changes in regulatory ECM proteins. By day 28, basement membrane proteins were reduced in drug-eluting stents in comparison with bare-metal stents. In contrast, the large aggregating proteoglycan aggrecan was increased. Aggrecanases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family contribute to the catabolism of vascular proteoglycans. An increase in ADAMTS-specific aggrecan fragments was accompanied by a notable shift from ADAMTS1 and ADAMTS5 to ADAMTS4 gene expression after stent implantation. Immunostaining in human stented coronary arteries confirmed the presence of aggrecan and aggrecan fragments, in particular, at the contacts of the stent struts with the artery. Further investigation of aggrecan presence in the human vasculature revealed that aggrecan and aggrecan cleavage were more abundant in human arteries than in human veins. In addition, aggrecan synthesis was induced on grafting a vein into the arterial circulation, suggesting an important role for aggrecan in vascular plasticity. Finally, lack of ADAMTS-5 activity in mice resulted in an accumulation of aggrecan and a dilation of the thoracic aorta, confirming that aggrecanase activity regulates aggrecan abundance in the arterial wall and contributes to vascular remodeling. CONCLUSIONS: Significant differences were identified by proteomics in the ECM of coronary arteries after bare-metal and drug-eluting stent implantation, most notably an upregulation of aggrecan, a major ECM component of cartilaginous tissues that confers resistance to compression. The accumulation of aggrecan coincided with a shift in ADAMTS gene expression. This study provides the first evidence implicating aggrecan and aggrecanases in the vascular injury response after stenting.
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Proteínas ADAMTS/metabolismo , Agrecanos , Vasos Coronarios/cirugía , Endopeptidasas/metabolismo , Matriz Extracelular/enzimología , Intervención Coronaria Percutánea/instrumentación , Proteómica/métodos , Stents , Remodelación Vascular , Proteínas ADAMTS/genética , Proteína ADAMTS5/genética , Proteína ADAMTS5/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Vasos Coronarios/enzimología , Vasos Coronarios/fisiopatología , Stents Liberadores de Fármacos , Endopeptidasas/genética , Femenino , Humanos , Masculino , Metales , Ratones Noqueados , Modelos Animales , Neointima , Diseño de Prótesis , Transducción de Señal , Sus scrofa , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8-/-) mice were generated. Fkbp8-/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.
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Apoptosis , Cardiotónicos/metabolismo , Retículo Endoplásmico/metabolismo , Corazón/fisiopatología , Hemodinámica , Estrés Fisiológico , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Aorta/patología , Apoptosis/efectos de los fármacos , Caspasa 12/metabolismo , Constricción Patológica , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Corazón/efectos de los fármacos , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hemodinámica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Ratones , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos , Presión , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión a Tacrolimus/deficiencia , Remodelación Ventricular/efectos de los fármacosRESUMEN
Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore-conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically-expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post-imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling.
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Núcleo Celular/metabolismo , Imagen Molecular/métodos , Transporte Activo de Núcleo Celular , Animales , Procesamiento de Imagen Asistido por Computador , Transporte de ProteínasRESUMEN
L-selectin is a cell adhesion molecule that tethers free-flowing leukocytes from the blood to luminal vessel walls, facilitating the initial stages of their emigration from the circulation toward an extravascular inflammatory insult. Following shear-resistant adhesion to the vessel wall, L-selectin has frequently been reported to be rapidly cleaved from the plasma membrane (known as ectodomain shedding), with little knowledge of the timing or functional consequence of this event. Using advanced imaging techniques, we observe L-selectin shedding occurring exclusively as primary human monocytes actively engage in transendothelial migration (TEM). Moreover, the shedding was localized to transmigrating pseudopods within the subendothelial space. By capturing monocytes in midtransmigration, we could monitor the subcellular distribution of L-selectin and better understand how ectodomain shedding might contribute to TEM. Mechanistically, L-selectin loses association with calmodulin (CaM; a negative regulator of shedding) specifically within transmigrating pseudopods. In contrast, L-selectin/CaM interaction remained intact in nontransmigrated regions of monocytes. We show phosphorylation of L-selectin at Ser 364 is critical for CaM dissociation, which is also restricted to the transmigrating pseudopod. Pharmacological or genetic inhibition of L-selectin shedding significantly increased pseudopodial extensions in transmigrating monocytes, which potentiated invasive behavior during TEM and prevented the establishment of front/back polarity for directional migration persistence once TEM was complete. We conclude that L-selectin shedding directly regulates polarity in transmigrated monocytes, which affirms an active role for this molecule in driving later stages of the multistep adhesion cascade.
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Polaridad Celular , Selectina L/metabolismo , Monocitos/citología , Secuencia de Aminoácidos , Adhesión Celular , Movimiento Celular , Citoplasma/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Leucocitos/metabolismo , Microscopía Electrónica de Transmisión , Microscopía por Video , Datos de Secuencia Molecular , Monocitos/metabolismo , Fosforilación , Serina/químicaRESUMEN
RATIONALE: Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. OBJECTIVE: Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. METHODS AND RESULTS: Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E-deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E-deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. CONCLUSIONS: We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.
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Aneurisma de la Aorta Abdominal/sangre , MicroARNs/fisiología , Anciano , Animales , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Biomarcadores/sangre , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/sangre , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patologíaRESUMEN
The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.
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Trimethylation of histone H3 lysine 9 and the subsequent binding of heterochromatin protein 1 (HP1) mediate the formation and maintenance of pericentromeric heterochromatin. Trimethylation of H3K9 is governed by the histone methyltransferase SUV39H1. Recent studies of HP1 dynamics revealed that HP1 is not a stable component of heterochromatin but is highly mobile (Cheutin, T., A.J. McNairn, T. Jenuwein, D.M. Gilbert, P.B. Singh, and T. Misteli. 2003. Science. 299:721-725; Festenstein, R., S.N. Pagakis, K. Hiragami, D. Lyon, A. Verreault, B. Sekkali, and D. Kioussis. 2003. Science. 299:719-721). Because the mechanism by which SUV39H1 is recruited to and interacts with heterochromatin is unknown, we studied the dynamic properties of SUV39H1 in living cells by using fluorescence recovery after photobleaching and fluorescence resonance energy transfer. Our results show that a substantial population of SUV39H1 is immobile at pericentromeric heterochromatin, suggesting that, in addition to its catalytic activity, SUV39H1 may also play a structural role at pericentromeric regions. Analysis of SUV39H1 deletion mutants indicated that the SET domain mediates this stable binding. Furthermore, our data suggest that the recruitment of SUV39H1 to heterochromatin is at least partly independent from that of HP1 and that HP1 transiently interacts with SUV39H1 at heterochromatin.
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Heterocromatina/metabolismo , Metiltransferasas/química , Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Catálisis/efectos de los fármacos , Línea Celular Tumoral , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN/efectos de los fármacos , Recuperación de Fluorescencia tras Fotoblanqueo , Transferencia Resonante de Energía de Fluorescencia , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas Luminiscentes/metabolismo , Ratones , Proteínas Mutantes/metabolismo , Células 3T3 NIH , Fenotipo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.
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Actomiosina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Movimiento Celular , Separación Celular , Humanos , Lamina Tipo A/metabolismo , Músculo Liso Vascular/citologíaRESUMEN
Many of the protein factors that play a role in nuclear export of mRNAs have been identified, but still little is known about how mRNAs are transported through the cell nucleus and which nuclear compartments are involved in mRNA transport. Using fluorescent 2'O-methyl oligoribonucleotide probes, we investigated the mobility of poly(A)+ RNA in the nucleoplasm and in nuclear speckles of U2OS cells. Quantitative analysis of diffusion using photobleaching techniques revealed that the majority of poly(A)+ RNA move throughout the nucleus, including in and out of speckles (also called SC-35 domains), which are enriched for splicing factors. Interestingly, in the presence of the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, the association of poly(A)+ RNA with speckles remained dynamic. Our results show that RNA movement is energy dependent and that the proportion of nuclear poly(A)+ RNA that resides in speckles is a dynamic population that transiently interacts with speckles independent of the transcriptional status of the cell. Rather than the poly(A)+ RNA within speckles serving a stable structural role, our findings support the suggestion of a more active role of these regions in nuclear RNA metabolism and/or transport.
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Núcleo Celular/metabolismo , Colorantes Fluorescentes/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética , Transporte Activo de Núcleo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Desoxiadenosinas/metabolismo , Diclororribofuranosil Benzoimidazol/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Proteínas Nucleares/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Rodaminas/química , Rodaminas/metabolismo , Factores de Empalme Serina-ArgininaRESUMEN
BACKGROUND: Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. ß-Adrenoceptor (ß-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of ß-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. METHODS AND RESULTS: A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the ß-AR agonist isoproterenol to induce ß1-AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. CONCLUSIONS: ß-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A-mediated phosphorylation of Ser279.
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Agonistas Adrenérgicos beta/farmacología , Núcleo Celular/efectos de los fármacos , Histona Desacetilasas/metabolismo , Isoproterenol/farmacología , Miocitos Cardíacos/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Receptores Adrenérgicos beta 1/efectos de los fármacos , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/enzimología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Histona Desacetilasas/genética , Masculino , Mutación , Miocitos Cardíacos/enzimología , Fosforilación , Unión Proteica , Proteína Fosfatasa 2/genética , Interferencia de ARN , Ratas Sprague-Dawley , Ratas Wistar , Receptores Adrenérgicos beta 1/metabolismo , Serina , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms. METHODS: We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies. RESULTS: Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study. CONCLUSION: The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery. FUNDING: UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Placa Aterosclerótica/metabolismo , Proteoma/metabolismo , Aterosclerosis/metabolismo , Biomarcadores/metabolismo , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/cirugía , Células Cultivadas , Endarterectomía Carotidea , Femenino , Humanos , Masculino , Miocitos del Músculo Liso/metabolismo , ProteómicaRESUMEN
Fluorescence in situ hybridization is a widely used technique in cell biology providing insight into the spatial organization of specific RNA transcripts in the cell nucleus. However, to further investigate the dynamics of the transcription process and the transport rates of RNAs through the nucleus, RNAs need to be visualized and tracked in the living cell. In past years, various methods have been developed with the aim of tagging specific RNAs with a fluorescent moiety without interfering with cell vitality. These methods include the delivery of probes into a living cell, the in vivo hybridization of fluorescent oligonucleotide probes to endogenous RNAs, and the microscopic imaging of the tagged RNAs in living cells. In this article, we review a number of methods for tagging and visualizing endogenous RNAs in living cells. In addition, a protocol is described that allows detection of various RNA types using fluorochrome-labeled 2(')-O-methyl oligoribonucleotide (2(')-OMe RNA) probes. Compared with conventional oligodeoxynucleotide probes, 2(')-OMe RNA probes are not degraded by nucleases, form stable hybrids with structured RNAs, and do not interfere with cell vitality.
Asunto(s)
Núcleo Celular/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Hibridación Fluorescente in Situ/métodos , ARN/análisis , Animales , Núcleo Celular/genética , HumanosRESUMEN
Chromosome ends are protected from degradation by the presence of the highly repetitive hexanucleotide sequence of TTAGGG and associated proteins. These so-called telomeric complexes are suggested to play an important role in establishing a functional nuclear chromatin organization. Using peptide nucleic acid (PNA) probes, we studied the dynamic behavior of telomeric DNA repeats in living human osteosarcoma U2OS cells. A fluorescent cy3-labeled PNA probe was introduced in living cells by glass bead loading and was shown to specifically associate with telomeric DNA shortly afterwards. Telomere dynamics were imaged for several hours using digital fluorescence microscopy. While the majority of telomeres revealed constrained diffusive movement, individual telomeres in a human cell nucleus showed significant directional movements. Also, a subfraction of telomeres were shown to associate and dissociate, suggesting that in vivo telomere clusters are not stable but dynamic structures. Furthermore, telomeres were shown to associate with promyelocytic leukemia (PML) bodies in a dynamic manner.
Asunto(s)
Ácidos Nucleicos de Péptidos/química , Telómero/fisiología , Telómero/ultraestructura , Secuencia de Bases , Neoplasias Óseas , Humanos , Cinética , Movimiento , Hibridación de Ácido Nucleico , Osteosarcoma , Secuencias Repetitivas de Ácidos Nucleicos , Células Tumorales CultivadasRESUMEN
The cellular processing of three fluorescent N, N'-bis(aminoalkyl)-1,4-diaminoanthraquinones (aminoalkyl=2-aminoethyl, 3-aminoprop-1-yl or 4-aminobut-1-yl) and their dinuclear platinum complexes in A2780 human ovarian carcinoma cells with acquired resistance to cisplatin has been monitored over time by time-lapse fluorescence microscopy. The results were compared with the previously reported observations in the parent A2780 cell line. The cellular distribution pattern for the free ligands is similar in sensitive and resistant cells, whereas significant differences in cellular distribution were observed in the case of the platinum complexes. In the cisplatin-resistant cell line the platinum complexes were found to be sequestrated in acidic vesicles in the cytosol from the very beginning of the incubation. This sequestration was not observed in the case of sensitive cells. Platinum accumulation in vesicles possibly presents a mechanism of resistance to platinum complexes. This mechanism appears to be unrelated to the mechanism of deactivation of platinum compounds by glutathione. Encapsulation of the dinuclear platinum complexes in lysosomal vesicles provides a plausible explanation for the decreased activity of these compounds in the resistant cell line, as compared to the sensitive cell line.
Asunto(s)
Resistencia a Antineoplásicos , Compuestos Organoplatinos/metabolismo , Compuestos Organoplatinos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Antraquinonas , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Transporte Biológico , Proliferación Celular/efectos de los fármacos , Cisplatino , Vesículas Citoplasmáticas/metabolismo , Femenino , Colorantes Fluorescentes , Humanos , Ligandos , Lisosomas/metabolismo , Microscopía Fluorescente , Compuestos Organoplatinos/química , Neoplasias Ováricas/patología , Relación Estructura-ActividadRESUMEN
PML bodies are nuclear organelles that are associated with various diseases and are suggested to be involved in multiple cellular activities including transcriptional regulation, apoptosis, and antiviral defence. Because many proteins with different functions aggregate in PML bodies, it has also been suggested that these bodies function as nuclear depots. Some proteins consistently found in PML bodies may form a stable scaffold that regulates the recruitment of other proteins. Thus, some proteins might be stably integrated into PML bodies while others continuously exchange with the nucleoplasm. To study the dynamic properties of PML bodies and resident proteins, we constructed fusion proteins of Sp100, PML, and CBP with autofluorescent proteins. Using time-lapse imaging, we show that PML bodies exhibit little movement but that small foci that contain Sp100 but not PML are dynamic and fuse with PML bodies. Furthermore, we show by monitoring fluorescence recovery after photobleaching that Sp100, PML, and CBP are dynamic components of PML bodies. This suggests that these proteins do not play a strict structural role in these bodies but that they function at other sites in the nucleoplasm.
Asunto(s)
Antígenos Nucleares/fisiología , Autoantígenos/fisiología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/fisiología , Factores de Transcripción/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Proteínas Nucleares/metabolismo , Plásmidos/metabolismo , Proteína de la Leucemia Promielocítica , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail. Purified Fin1p interacts with recombinant yeast 14-3-3 proteins. This interaction is strongly reduced after dephosphorylation of Fin1p. Surface plasmon resonance analysis showed that Fin1p has a higher affinity for Bmh2p than for Bmh1p (K(D) 289 versus 585 nm). Sequences in both the central and C-terminal part of Fin1p are required for the interaction with Bmh2p in the two-hybrid system. In yeast strains lacking 14-3-3 proteins Fin1 filament formation was observed, indicating that the 14-3-3 proteins are not required for this process. Fin1 also interacts with itself in the two-hybrid system. For this interaction sequences at the C terminus, containing one of two putative coiled-coil regions, are sufficient. Fin1p-Fin1p interactions were demonstrated in vivo by fluorescent resonance energy transfer between cyan fluorescent protein-labeled Fin1p and yellow fluorescent protein-labeled Fin1p.