RESUMO
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.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Frequência Cardíaca , Ativação do Canal Iônico , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miocárdio/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Camundongos , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/genética , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismoRESUMO
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.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Metabolômica/métodos , Fluxo de Trabalho , Animais , Coração/fisiologia , Camundongos , Miocárdio/metabolismo , Padrões de ReferênciaRESUMO
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.
Assuntos
Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Proteínas ras/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Cardiomegalia/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Insuficiência Cardíaca/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas ras/genéticaRESUMO
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.
Assuntos
Hipocampo/fisiologia , Neurogênese , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Proteínas ras/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas ras/genéticaRESUMO
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.
Assuntos
Envelhecimento/metabolismo , Insuficiência Cardíaca/prevenção & controle , Miocárdio/enzimologia , Sístole , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda , Proteínas ras/deficiência , Adaptação Fisiológica , Fatores Etários , Envelhecimento/genética , Animais , Sinalização do Cálcio , Débito Cardíaco , Progressão da Doença , Genótipo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/patologia , Fenótipo , Retículo Sarcoplasmático/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologia , Proteínas ras/genéticaRESUMO
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.
Assuntos
Canais de Cálcio Tipo L , Miócitos Cardíacos , Animais , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Camundongos , Miócitos Cardíacos/metabolismo , Membrana Celular/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Camundongos Transgênicos , Proteínas rasRESUMO
The molecular mechanisms governing the spontaneous recovery seen following brain injury remain elusive, but recent studies indicate that injury-induced stimulation of hippocampal neurogenesis contributes to the repair process. The therapeutic potential of endogenous neurogenesis is tempered by the demonstration that traumatic brain injury (TBI) results in the selective death of adult-born immature neurons, compromising the cell population poised to compensate for trauma-induced neuronal loss. Here, we identify the Ras-related GTPase, Rit, as a critical player in the survival of immature hippocampal neurons following brain injury. While Rit knock-out (Rit(-/-)) did not alter hippocampal development, hippocampal neural cultures derived from Rit(-/-) mice display increased cell death and blunted MAPK cascade activation in response to oxidative stress, without affecting BDNF-dependent signaling. When compared with wild-type hippocampal cultures, Rit loss rendered immature (Dcx(+)) neurons susceptible to oxidative damage, without altering the survival of neural progenitor (Nestin(+)) cells. Oxidative stress is a major contributor to neuronal cell death following brain injury. Consistent with the enhanced vulnerability of cultured Rit(-/-) immature neurons, Rit(-/-) mice exhibited a significantly greater loss of adult-born immature neurons within the dentate gyrus after TBI. In addition, post-TBI neuronal remodeling was blunted. Together, these data identify a new and unexpected role for Rit in injury-induced neurogenesis, functioning as a selective survival mechanism for immature hippocampal neurons within the subgranular zone of the dentate gyrus following TBI.
Assuntos
Sobrevivência Celular/fisiologia , Hipocampo/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Dendritos/metabolismo , Proteína Duplacortina , Hipocampo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Estresse Oxidativo/fisiologia , Proteínas ras/genéticaRESUMO
The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and Bcl(XL) proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Estresse Fisiológico/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transcrição Gênica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas ras/genéticaRESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A, progerin, that terminates with a farnesylcysteine. HGPS knock-in mice (Lmna(HG/+)) develop severe progeria-like disease phenotypes. These phenotypes can be ameliorated with a protein farnesyltransferase inhibitor (FTI), suggesting that progerin's farnesyl lipid is important for disease pathogenesis and raising the possibility that FTIs could be useful for treating humans with HGPS. Subsequent studies showed that mice expressing non-farnesylated progerin (Lmna(nHG/+) mice, in which progerin's carboxyl-terminal -CSIM motif was changed to -SSIM) also develop severe progeria, raising doubts about whether any treatment targeting protein prenylation would be particularly effective. We suspected that those doubts might be premature and hypothesized that the persistent disease in Lmna(nHG/+) mice could be an unanticipated consequence of the cysteine-to-serine substitution that was used to eliminate farnesylation. To test this hypothesis, we generated a second knock-in allele yielding non-farnesylated progerin (Lmna(csmHG)) in which the carboxyl-terminal -CSIM motif was changed to -CSM. We then compared disease phenotypes in mice harboring the Lmna(nHG) or Lmna(csmHG) allele. As expected, Lmna(nHG/+) and Lmna(nHG/nHG) mice developed severe progeria-like disease phenotypes, including osteolytic lesions and rib fractures, osteoporosis, slow growth and reduced survival. In contrast, Lmna(csmHG/+) and Lmna(csmHG/csmHG) mice exhibited no bone disease and displayed entirely normal body weights and survival. The frequencies of misshapen cell nuclei were lower in Lmna(csmHG/+) and Lmna(csmHG/csmHG) fibroblasts. These studies show that the ability of non-farnesylated progerin to elicit disease depends on the carboxyl-terminal mutation used to eliminate protein prenylation.
Assuntos
Farnesiltranstransferase/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progéria/genética , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Substituição de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Feminino , Técnicas de Introdução de Genes , Imidazóis/farmacologia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Masculino , Camundongos , Mutação , Proteínas Nucleares/química , Fenótipo , Progéria/metabolismo , Progéria/patologia , Progéria/fisiopatologia , Precursores de Proteínas/química , Prenilação de ProteínaRESUMO
The L-type calcium channel (LTCC) provides trigger Ca(2+) for sarcoplasmic reticulum Ca-release, and LTCC function is influenced by interacting proteins including the LTCC distal COOH terminus (DCT) and calmodulin. DCT is proteolytically cleaved and reassociates with the LTCC complex to regulate calcium channel function. DCT reduces LTCC barium current (I(Ba,L)) in reconstituted channel complexes, yet the contribution of DCT to LTCC Ca(2+) current (I(Ca,L)) in cardiomyocyte systems is unexplored. This study tests the hypothesis that DCT attenuates cardiomyocyte I(Ca,L). We measured LTCC current and Ca(2+) transients with DCT coexpressed in murine cardiomyocytes. We also heterologously coexpressed DCT and Ca(V)1.2 constructs with truncations corresponding to the predicted proteolytic cleavage site, Ca(V)1.2Δ1801, and a shorter deletion corresponding to well-studied construct, Ca(V)1.2Δ1733. DCT inhibited I(Ba,L) in cardiomyocytes, and in human embryonic kidney (HEK) 293 cells expressing Ca(V)1.2Δ1801 and Ca(V)1.2Δ1733. Ca(2+)-CaM relieved DCT block in cardiomyocytes and HEK cells. The selective block of I(Ba,L) combined with Ca(2+)-CaM effects suggested that DCT-mediated blockade may be relieved under conditions of elevated Ca(2+). We therefore tested the hypothesis that DCT block is dynamic, increasing under relatively low Ca(2+), and show that DCT reduced diastolic Ca(2+) at low stimulation frequencies but spared high frequency Ca(2+) entry. DCT reduction of diastolic Ca(2+) and relief of block at high pacing frequencies and under conditions of supraphysiological bath Ca(2+) suggests that a physiological function of DCT is to increase the dynamic range of Ca(2+) transients in response to elevated pacing frequencies. Our data motivate the new hypothesis that DCT is a native reverse use-dependent inhibitor of LTCC current.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Coração/fisiologia , Animais , Bário/farmacologia , Cálcio/farmacologia , Cálcio/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Estimulação Cardíaca Artificial , Fenômenos Eletrofisiológicos , Feminino , Células HEK293 , Humanos , Cinética , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , GravidezRESUMO
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.
RESUMO
Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca(1)a(2)X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca(1)a(2)X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors.
Assuntos
Alquil e Aril Transferases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Alquilação , Catálise , Cinética , Espectroscopia de Ressonância Magnética , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Especificidade por SubstratoRESUMO
Protein farnesyltransferase (FTase) inhibitors, generally called "FTIs," block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis-GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl-prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl-prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Metaloendopeptidases/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Peptidomiméticos/farmacologia , Inibidores de Proteases/farmacologia , Precursores de Proteínas/metabolismo , Animais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lamina Tipo A , CamundongosRESUMO
Lamin A is formed from prelamin A by four post-translational processing steps-farnesylation, release of the last three amino acids of the protein, methylation of the farnesylcysteine and the endoproteolytic release of the C-terminal 15 amino acids (including the farnesylcysteine methyl ester). When the final processing step does not occur, a farnesylated and methylated prelamin A accumulates in cells, causing a severe progeroid disease, restrictive dermopathy (RD). Whether RD is caused by the retention of farnesyl lipid on prelamin A, or by the retention of the last 15 amino acids of the protein, is unknown. To address this issue, we created knock-in mice harboring a mutant Lmna allele (LmnanPLAO) that yields exclusively non-farnesylated prelamin A (and no lamin C). These mice had no evidence of progeria but succumbed to cardiomyopathy. We suspected that the non-farnesylated prelamin A in the tissues of these mice would be strikingly mislocalized to the nucleoplasm, but this was not the case; most was at the nuclear rim (indistinguishable from the lamin A in wild-type mice). The cardiomyopathy could not be ascribed to an absence of lamin C because mice expressing an otherwise identical knock-in allele yielding only wild-type prelamin A appeared normal. We conclude that lamin C synthesis is dispensable in mice and that the failure to convert prelamin A to mature lamin A causes cardiomyopathy (at least in the absence of lamin C). The latter finding is potentially relevant to the long-term use of protein farnesyltransferase inhibitors, which lead to an accumulation of non-farnesylated prelamin A.
Assuntos
Cardiomiopatias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Progéria/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Animais , Cardiomiopatias/etiologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas de Introdução de Genes , Lamina Tipo A/metabolismo , Camundongos , Camundongos Endogâmicos , Modelos Animais , Progéria/etiologia , Prenilação de ProteínaRESUMO
Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply 'switch' upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Antígenos CD , Caderinas/genética , Caderinas/metabolismo , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Cinetocoros/metabolismo , Camundongos , Microscopia , Prenilação , Interferência de RNA , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Prenylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as Ras. Methods allowing rapid and selective detection of protein farnesylation and geranylgeranylation are fundamental for the understanding of prenylated protein function and for monitoring efficacy of drugs such as farnesyltransferase inhibitors (FTIs). Although the natural substrates for prenyltransferases are farnesyl pyrophosphate and geranylgeranyl pyrophosphate, farnesyltransferase has been shown to incorporate isoprenoid analogues into protein substrates. In this study, protein prenyltransferase targets were labeled using anilinogeraniol, the alcohol precursor to the unnatural farnesyl pyrophosphate analogue 8-anilinogeranyl diphosphate in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect the anilinogeranyl-modified proteins. Coupling this highly effective labeling/detection method with two-dimensional electrophoresis and subsequent Western blotting allowed simple, rapid analysis of the complex farnesylated proteome. For example, this method elucidated the differential effects induced by two chemically distinct FTIs, BMS-214,662 and L-778,123. Although both FTIs strongly inhibited farnesylation of many proteins such as Lamins, NAP1L1, N-Ras, and H-Ras, only the dual prenylation inhibitor L-778,123 blocked prenylation of Pex19, RhoB, K-Ras, Cdc42, and Rap1. This snapshot approach has significant advantages over traditional techniques, including radiolabeling, anti-farnesyl antibodies, or mass spectroscopy, and enables dynamic analysis of the farnesylated proteome.
Assuntos
Prenilação de Proteína , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Coloração e Rotulagem/métodos , Compostos de Anilina/farmacologia , Western Blotting/métodos , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Farnesiltranstransferase/metabolismo , Células HL-60 , Humanos , Imidazóis/farmacologia , Células K562 , Modelos Biológicos , Especificidade por SubstratoRESUMO
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.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Sarcoma de Ewing , Masculino , Humanos , Animais , Camundongos , Criança , Sarcoma de Ewing/patologia , Glicogênio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Hutchinson-Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5' portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5' to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease 'HGPS splicing'. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells.
Assuntos
Mutação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/uso terapêutico , Progéria/genética , Progéria/terapia , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Processamento Alternativo , Sequência de Bases , Linhagem Celular , Células Cultivadas , Éxons , Fibroblastos/metabolismo , Terapia Genética , Humanos , Lamina Tipo A , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Progéria/metabolismoRESUMO
Hutchinson-Gilford progeria syndrome (HGPS), a rare disease that results in what appears to be premature aging, is caused by the production of a mutant form of prelamin A known as progerin. Progerin retains a farnesyl lipid anchor at its carboxyl terminus, a modification that is thought to be important in disease pathogenesis. Inhibition of protein farnesylation improves the hallmark nuclear shape abnormalities in HGPS cells and ameliorates disease phenotypes in mice harboring a knockin HGPS mutation (LmnaHG/+). The amelioration of disease, however, is incomplete, leading us to hypothesize that nonfarnesylated progerin also might be capable of eliciting disease. To test this hypothesis, we created knockin mice expressing nonfarnesylated progerin (LmnanHG/+). LmnanHG/+ mice developed the same disease phenotypes observed in LmnaHG/+ mice, although the phenotypes were milder, and mouse embryonic fibroblasts (MEFs) derived from these mice contained fewer misshapen nuclei. The steady-state levels of progerin in LmnanHG/+ MEFs and tissues were lower, suggesting a possible explanation for the milder phenotypes. These data support the concept that inhibition of protein farnesylation in progeria could be therapeutically useful but also suggest that this approach may be limited, as progerin elicits disease phenotypes whether or not it is farnesylated.