RESUMEN
L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel ß subunits (CaVß) to promote inhibition of ICa,L. In addition to CaVß interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to ß-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.
Asunto(s)
Canales de Calcio Tipo L , Miocitos Cardíacos , Animales , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/genética , Ratones , Miocitos Cardíacos/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Ratones Transgénicos , Proteínas rasRESUMEN
Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. Calcium (Ca2+) is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC), but not other Ca2+ regulatory proteins (SERCA or RYR), induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD induces the cell cycle in CMs by modulating calcineurin activity and translocating Hoxb13 to the CM nucleus. Together, this represents a robust pathway for regenerative strategies.
RESUMEN
Oligodendrocytes (OLs) generate lipid-rich myelin membranes that wrap axons to enable efficient transmission of electrical impulses. Using a RIT1 knockout mouse model and in situ high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) coupled with MS-based lipidomic analysis to determine the contribution of RIT1 to lipid homeostasis. Here, we report that RIT1 loss is associated with altered lipid levels in the central nervous system (CNS), including myelin-associated lipids within the corpus callosum (CC). Perturbed lipid metabolism was correlated with reduced numbers of OLs, but increased numbers of GFAP+ glia, in the CC, but not in grey matter. This was accompanied by reduced myelin protein expression and axonal conduction deficits. Behavioral analyses revealed significant changes in voluntary locomotor activity and anxiety-like behavior in RIT1KO mice. Together, these data reveal an unexpected role for RIT1 in the regulation of cerebral lipid metabolism, which coincide with altered white matter tract oligodendrocyte levels, reduced axonal conduction velocity, and behavioral abnormalities in the CNS.
RESUMEN
Glycogen dysregulation is a hallmark of aging, and aberrant glycogen drives metabolic reprogramming and pathogenesis in multiple diseases. However, glycogen heterogeneity in healthy and diseased tissues remains largely unknown. Herein, we describe a method to define spatial glycogen architecture in mouse and human tissues using matrix-assisted laser desorption/ionization mass spectrometry imaging. This assay provides robust and sensitive spatial glycogen quantification and architecture characterization in the brain, liver, kidney, testis, lung, bladder, and even the bone. Armed with this tool, we interrogated glycogen spatial distribution and architecture in different types of human cancers. We demonstrate that glycogen stores and architecture are heterogeneous among diseases. Additionally, we observe unique hyperphosphorylated glycogen accumulation in Ewing sarcoma, a pediatric bone cancer. Using preclinical models, we correct glycogen hyperphosphorylation in Ewing sarcoma through genetic and pharmacological interventions that ablate in vivo tumor growth, demonstrating the clinical therapeutic potential of targeting glycogen in Ewing sarcoma.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Sarcoma de Ewing , Masculino , Humanos , Animales , Ratones , Niño , Sarcoma de Ewing/patología , Glucógeno , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to ß-adrenergic receptor (ß-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of ß-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of ß-AR stimulation, we crossed a ß1,ß2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express ß1,ß2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of ß1,ß2-AR signaling and that Rad deletion supersedes ß-AR signaling to the LTCC to enhance in vivo heart function.
Asunto(s)
Calcio , Miocitos Cardíacos , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Isoproterenol/farmacología , Ratones , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMEN
Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of ß-adrenergic receptor signaling cascade (ß-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of ß-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in ß-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which ß-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with ß-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad - LTCC can thus elevate HR while retaining ßAR responsiveness.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Frecuencia Cardíaca , Activación del Canal Iónico , Proteínas de Unión al GTP Monoméricas/metabolismo , Miocardio/metabolismo , Animales , Canales de Calcio Tipo L/genética , Ratones , Ratones Transgénicos , Proteínas de Unión al GTP Monoméricas/genética , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismoAsunto(s)
Encéfalo/metabolismo , Gangliósidos/metabolismo , Espectrometría de Masas , Imagen Molecular , Animales , RatonesRESUMEN
MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS-based metabolomics workflow that uses insoluble protein-derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute ß-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolómica/métodos , Flujo de Trabajo , Animales , Corazón/fisiología , Ratones , Miocardio/metabolismo , Estándares de ReferenciaRESUMEN
Existing therapies to improve heart function target ß-adrenergic receptor (ß-AR) signaling and Ca2+ handling and often lead to adverse outcomes. This underscores an unmet need for positive inotropes that improve heart function without any adverse effects. The GTPase Ras associated with diabetes (RAD) regulates L-type Ca2+ channel (LTCC) current (ICa,L). Global RAD-knockout mice (gRAD-/-) have elevated Ca2+ handling and increased cardiac hypertrophy, but RAD is expressed also in noncardiac tissues, suggesting the possibility that pathological remodeling is due also to noncardiac effects. Here, we engineered a myocardial-restricted inducible RAD-knockout mouse (RADΔ/Δ). Using an array of methods and techniques, including single-cell electrophysiological and calcium transient recordings, echocardiography, and radiotelemetry monitoring, we found that RAD deficiency results in a sustained increase of inotropy without structural or functional remodeling of the heart. ICa,L was significantly increased, with RAD loss conferring a ß-AR-modulated phenotype on basal ICa,L Cardiomyocytes from RADΔ/Δ hearts exhibited enhanced cytosolic Ca2+ handling, increased contractile function, elevated sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2a) expression, and faster lusitropy. These results argue that myocardial RAD ablation promotes a beneficial elevation in Ca2+ dynamics, which would obviate a need for increased ß-AR signaling to improve cardiac function.
Asunto(s)
Contracción Miocárdica/fisiología , Miocardio/metabolismo , Proteínas ras/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/fisiología , Señalización del Calcio/fisiología , Cardiomegalia/metabolismo , GTP Fosfohidrolasas/metabolismo , Insuficiencia Cardíaca/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Proteínas ras/genéticaRESUMEN
The protein Rad interacts with the LTCC to modulate trigger Ca2+, hence to govern contractility. Reducing Rad levels increases cardiac output. Ablation of Rad also attenuated the inflammatory response following acute myocardial infarction (AMI). Future studies to target deletion of Rad in the heart could be conducted to establish a novel treatment paradigm whereby pathologically stressed hearts would be given a safe, stable positive inotropic support without arrhythmias and without pathological structural remodeling. Future investigations will also focus on establishing inhibitors of Rad, and testing the efficacy of Rad-deletion in cardioprotection relative to the time of onset of AMI.
RESUMEN
PURPOSE: Ras-like without CAAX 1 (RIT1/Rit) is a member of the Ras subfamily of small GTP-binding proteins with documented roles in regulating neuronal function, including contributions to neurotrophin signaling, neuronal survival, and neurogenesis. The aim of the study was to (1) examine the expression of RIT1 protein in mouse retina and retinal cell types and (2) determine whether RIT1 contributes to retinal ganglion cell (RGC) survival and synaptic stability following excitotoxic stress. MATERIALS AND METHODS: Gene expression and immunohistochemical analysis were used to examine RIT1 expression in the mouse retina. Primary RGC and Müller glia cultures were used to validate novel RIT1 lentiviral RNAi silencing reagents, and to demonstrate that RIT1 loss does not alter RGC morphology. Finally, in vitro glutamate exposure identified a role for RIT1 in the adaptation of RGCs to excitotoxic stress. RESULTS: Gene expression analysis and immunohistochemical studies in whole eyes and primary cell culture demonstrate RIT1 expression throughout the retina, including Müller glia and RGCs. While genetic RIT1 knockout (RIT1-KO) does not affect gross retinal anatomy, including the thickness of constituent retinal layers or RGC cell numbers, RNAi-mediated RIT1 silencing results in increased RGC death and synaptic loss following exposure to excitotoxic stress. CONCLUSIONS: RIT1 is widely expressed in the murine retina, including both Müller glia and RGCs. While genetic deletion of RIT1 does not result in gross retinal abnormalities, these studies identify a novel role for RIT1 in the adaptation of RGC to excitotoxic stress, with RIT1 promoting both neuronal survival and the retention of PSD-95+ synapses.
Asunto(s)
Apoptosis , Regulación de la Expresión Génica , ARN/genética , Células Ganglionares de la Retina/metabolismo , Proteínas ras/genética , Animales , Supervivencia Celular , Células Cultivadas , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Modelos Animales , Reacción en Cadena de la Polimerasa , Células Ganglionares de la Retina/citología , Transducción de Señal , Proteínas ras/biosíntesisRESUMEN
The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2+ channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad-/- calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, +11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.
Asunto(s)
Adipogénesis/fisiología , Densidad Ósea/fisiología , Médula Ósea/enzimología , Osteogénesis/fisiología , Proteínas ras/metabolismo , Tejido Adiposo/patología , Animales , Médula Ósea/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Insulin-like growth factor 1 (IGF-1) is known to have diverse effects on brain structure and function, including the promotion of stem cell proliferation and neurogenesis in the adult dentate gyrus. However, the intracellular pathways downstream of the IGF-1 receptor that contribute to these diverse physiological actions remain relatively uncharacterized. Here, we demonstrate that the Ras-related GTPase, RIT1, plays a critical role in IGF-1-dependent neurogenesis. Studies in hippocampal neuronal precursor cells (HNPCs) demonstrate that IGF-1 stimulates a RIT1-dependent increase in Sox2 levels, resulting in pro-neural gene expression and increased cellular proliferation. In this novel cascade, RIT1 stimulates Akt-dependent phosphorylation of Sox2 at T118, leading to its stabilization and transcriptional activation. When compared to wild-type HNPCs, RIT1 -/- HNPCs show deficient IGF-1-dependent Akt signaling and neuronal differentiation, and accordingly, Sox2-dependent hippocampal neurogenesis is significantly blunted following IGF-1 infusion in knockout (RIT1 -/- ) mice. Consistent with a role for RIT1 function in the modulation of activity-dependent plasticity, exercise-mediated potentiation of hippocampal neurogenesis is also diminished in RIT1 -/- mice. Taken together, these data identify the previously uncharacterized IGF1-RIT1-Akt-Sox2 signaling pathway as a key component of neurogenic niche sensing, contributing to the regulation of neural stem cell homeostasis.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Neurogénesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción SOXB1/metabolismo , Proteínas ras/metabolismo , Animales , Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Noqueados , Modelos Biológicos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Fosforilación , Condicionamiento Físico Animal , Células Piramidales/citología , Células Piramidales/metabolismo , Interferencia de ARN , Factores de Transcripción SOXB1/genética , Transducción de Señal/efectos de los fármacos , Proteínas ras/genéticaRESUMEN
Adult neurogenesis, the process of generating mature neurons from neuronal progenitor cells, makes critical contributions to neural circuitry and brain function in both healthy and disease states. Neurogenesis is a highly regulated process in which diverse environmental and physiological stimuli are relayed to resident neural stem cell populations to control the transcription of genes involved in self-renewal and differentiation. Understanding the molecular mechanisms governing neurogenesis is necessary for the development of translational strategies to harness this process for neuronal repair. Here we report that the Ras-related GTPase RIT1 serves to control the sequential proliferation and differentiation of adult hippocampal neural progenitor cells, with in vivo expression of active RIT1 driving robust adult neurogenesis. Gene expression profiling analysis demonstrates increased expression of a specific set of transcription factors known to govern adult neurogenesis in response to active RIT1 expression in the hippocampus, including sex-determining region Y-related HMG box 2 (Sox2), a well established regulator of stem cell self-renewal and neurogenesis. In adult hippocampal neuronal precursor cells, RIT1 controls an Akt-dependent signaling cascade, resulting in the stabilization and transcriptional activation of phosphorylated Sox2. This study supports a role for RIT1 in relaying niche-derived signals to neural/stem progenitor cells to control transcription of genes involved in self-renewal and differentiation.
Asunto(s)
Hipocampo/fisiología , Neurogénesis , Factores de Transcripción SOXB1/metabolismo , Transcripción Genética , Proteínas ras/metabolismo , Animales , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas ras/genéticaRESUMEN
Sympathetic stimulation modulates L-type calcium channel (LTCC) gating to contribute to increased systolic heart function. Rad is a monomeric G-protein that interacts with LTCC. Genetic deletion of Rad (Rad-/-) renders LTCC in a sympathomimetic state. The study goal was to use a clinically inspired pharmacological stress echocardiography test, including analysis of global strain, to determine whether Rad-/- confers tonic positive inotropic heart function. Sarcomere dynamics and strain showed partial parallel isoproterenol (ISO) responsiveness for wild-type (WT) and for Rad-/-. Rad-/- basal inotropy was elevated compared to WT but was less responsiveness to ISO. Rad protein levels were lower in human patients with end-stage non-ischemic heart failure. These results show that Rad reduction provides a stable inotropic response rooted in sarcomere level function. Thus, reduced Rad levels in heart failure patients may be a compensatory response to need for increased output in the setting of HF. Rad deletion suggests a future therapeutic direction for inotropic support.
Asunto(s)
Cardiomegalia/metabolismo , Eliminación de Gen , Frecuencia Cardíaca , Corazón/inervación , Contracción Miocárdica , Sistema Nervioso Simpático/fisiopatología , Proteínas ras/deficiencia , Animales , Canales de Calcio Tipo L/metabolismo , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Estudios de Casos y Controles , Ecocardiografía de Estrés/métodos , Genotipo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Isoproterenol/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Fenotipo , Sarcómeros/metabolismo , Sarcómeros/patología , Sistema Nervioso Simpático/efectos de los fármacos , Simpatomiméticos/administración & dosificación , Remodelación Ventricular , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
Rad-GTPase is a regulator of L-type calcium current (LTCC), with increased calcium current observed in Rad knockout models. While mouse models that result in elevated LTCC have been associated with heart failure, our laboratory and others observe a hypercontractile phenotype with enhanced calcium homeostasis in Rad(-/-). It is currently unclear whether this observation represents an early time point in a decompensatory progression towards heart failure or whether Rad loss drives a novel phenotype with stable enhanced function. We test the hypothesis that Rad(-/-) drives a stable nonfailing hypercontractile phenotype in adult hearts, and we examine compensatory regulation of sarcoplasmic reticulum (SR) loading and protein changes. Heart function was measured in vivo with echocardiography. In vivo heart function was significantly improved in adult Rad(-/-) hearts compared with wild type. Heart wall dimensions were significantly increased, while heart size was decreased, and cardiac output was not changed. Cardiac function was maintained through 18 mo of age with no decompensation. SR releasable Ca(2+) was increased in isolated Rad(-/-) ventricular myocytes. Higher Ca(2+) load was accompanied by sarco/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) protein elevation as determined by immunoblotting and a rightward shift in the thapsigargan inhibitor-response curve. Rad(-/-) promotes morphological changes accompanied by a stable increase in contractility with aging and preserved cardiac output. The Rad(-/-) phenotype is marked by enhanced systolic and diastolic function with increased SR uptake, which is consistent with a model that does not progress into heart failure.
Asunto(s)
Envejecimiento/metabolismo , Insuficiencia Cardíaca/prevención & control , Miocardio/enzimología , Sístole , Disfunción Ventricular Izquierda/enzimología , Función Ventricular Izquierda , Proteínas ras/deficiencia , Adaptación Fisiológica , Factores de Edad , Envejecimiento/genética , Animales , Señalización del Calcio , Gasto Cardíaco , Progresión de la Enfermedad , Genotipo , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/patología , Fenotipo , Retículo Sarcoplasmático/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatología , Proteínas ras/genéticaRESUMEN
As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids.
Asunto(s)
Alcaloides/metabolismo , Transferasas Alquil y Aril/metabolismo , Compuestos Macrocíclicos/metabolismo , Nicotiana/enzimología , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/metabolismo , Alcaloides/química , Transferasas Alquil y Aril/química , Compuestos Macrocíclicos/química , Modelos Moleculares , Fosfatos de Poliisoprenilo/química , Sesquiterpenos/química , Terpenos/química , Terpenos/metabolismo , Nicotiana/química , Nicotiana/metabolismoRESUMEN
The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.
Asunto(s)
Proteínas Portadoras/metabolismo , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Cinetocoros/efectos de los fármacos , Mitosis/efectos de los fármacos , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/efectos de los fármacos , Complejo Dinactina , Dineínas/efectos de los fármacos , Farnesiltransferasa/metabolismo , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Prenilación de Proteína , XenopusRESUMEN
Rit, a member of the Ras family of GTPases, has been shown to promote cell survival in response to oxidative stress, in part by directing an evolutionarily conserved p38 MAPK-Akt survival cascade. Aberrant Rit signaling has recently been implicated as a driver mutation in human cancer, adding importance to the characterization of critical Rit effector pathways. However, the mechanism by which Rit-p38 signaling regulated Akt activity was unknown. Here, we identify mTORC2 as a critical downstream mediator of Rit-dependent survival signaling in response to reactive oxygen species (ROS) stress. Rit interacts with Sin1 (MAPKAP1), and Rit loss compromises ROS-dependent mTORC2 complex activation, blunting mTORC2-mediated phosphorylation of Akt kinase. Taken together, our findings demonstrate that the p38/mTORC2/Akt signaling cascade mediates Rit-dependent oxidative stress survival. Inhibition of this previously unrecognized cascade should be explored as a potential therapy of Rit-dependent malignancies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos Multiproteicos/metabolismo , Estrés Oxidativo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas ras/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Fosforilación , Transducción de SeñalRESUMEN
Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA's clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the clinical properties of VPA. VPA can be used experimentally to down regulate DICER protein levels, which likely reflects a natural regulation of DICER.