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1.
Mol Cell ; 83(21): 3904-3920.e7, 2023 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-37879334

RESUMEN

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.


Asunto(s)
Metabolismo Energético , Ácido Láctico , Ácido Láctico/metabolismo , Transporte de Electrón , Fosforilación Oxidativa , Glucólisis/fisiología , Adenosina Trifosfato/metabolismo
2.
Mol Cell ; 79(6): 934-949.e14, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32822587

RESUMEN

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.


Asunto(s)
ADP-Ribosilación/genética , Adipogénesis/genética , Histonas/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Adenosina Difosfato Ribosa/genética , Adipocitos/metabolismo , Adipocitos/patología , Animales , Línea Celular , Daño del ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Fosforilación/genética , ARN Nucleolar Pequeño/genética
3.
Mol Cell ; 65(2): 260-271, 2017 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-28107648

RESUMEN

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPß, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPß's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPß from PARylation-mediated inhibition. This promotes the binding of C/EBPß at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPß, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes.


Asunto(s)
Adipocitos/enzimología , Adipogénesis , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Madre Embrionarias/enzimología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Procesamiento Proteico-Postraduccional , Transcripción Genética , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/genética , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/efectos de los fármacos , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células 3T3 NIH , Fenotipo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica , Dominios Proteicos , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Activación Transcripcional , Transfección
4.
Mol Cell ; 63(3): 349-51, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27494555

RESUMEN

In a recent issue of Science, Wright et al. (2016) describe a pathway for the synthesis of nuclear ATP, leading from NAD(+) to poly(ADP-ribose) to ADP-ribose to ATP, which supports the activity of ATP-dependent chromatin remodeling enzymes during hormone-dependent transcription.


Asunto(s)
Adenosina Difosfato Ribosa , Poli(ADP-Ribosa) Polimerasas , Adenosina Trifosfato , Núcleo Celular , NAD , Poli Adenosina Difosfato Ribosa
5.
Mol Cell ; 59(4): 651-63, 2015 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-26236015

RESUMEN

Hydrogen peroxide (H2O2) released from mitochondria regulates various cell signaling pathways. Given that H2O2-eliminating enzymes such as peroxiredoxin III (PrxIII) are abundant in mitochondria, however, it has remained unknown how such release can occur. Active PrxIII-SH undergoes reversible inactivation via hyperoxidation to PrxIII-SO2, which is then reduced by sulfiredoxin. We now show that the amounts of PrxIII-SO2 and sulfiredoxin undergo antiphasic circadian oscillation in the mitochondria of specific tissues of mice maintained under normal conditions. Cytosolic sulfiredoxin was found to be imported into the mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is promoted by H2O2 released from mitochondria. The imported sulfiredoxin is degraded by Lon in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of sulfiredoxin provide the basis for sulfiredoxin oscillation and consequent PrxIII-SO2 oscillation in mitochondria and likely result in an oscillatory H2O2 release.


Asunto(s)
Ritmo Circadiano , Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Animales , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Oxidación-Reducción , Peroxiredoxina III/metabolismo , Proteasa La/metabolismo , Transporte de Proteínas , Proteolisis , Dióxido de Azufre/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo
6.
Mol Cell ; 46(5): 584-94, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22681886

RESUMEN

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.


Asunto(s)
Glándulas Suprarrenales/metabolismo , Retroalimentación Fisiológica , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Peroxiredoxina III/metabolismo , Animales , Colesterol/metabolismo , Corticosterona/biosíntesis , Ratones , Ratones Transgénicos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxiredoxina III/fisiología , Fosfoproteínas/metabolismo , Fosforilación , Esteroide 11-beta-Hidroxilasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
7.
J Biol Chem ; 287(15): 12405-16, 2012 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-22334709

RESUMEN

NMNAT-1 and PARP-1, two key enzymes in the NAD(+) metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters where it produces NAD(+) to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independently of NAD(+) production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD(+) in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent poly(ADP-ribosyl)ation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters.


Asunto(s)
Regulación de la Expresión Génica , Nicotinamida-Nucleótido Adenililtransferasa/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiología , Regiones Promotoras Genéticas , Transporte Activo de Núcleo Celular , Línea Celular , Activación Enzimática , Perfilación de la Expresión Génica , Humanos , NAD/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética
9.
Science ; 372(6545): 968-972, 2021 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-33888598

RESUMEN

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.


Asunto(s)
Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Prolina/biosíntesis , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Ciclo del Ácido Cítrico , Colágeno/metabolismo , Medios de Cultivo , Citosol/metabolismo , Femenino , Ácido Fólico/metabolismo , Técnicas de Inactivación de Genes , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Humanos , Metaboloma , Ratones , Ratones Desnudos , Proteínas Mitocondriales/genética , Estrés Oxidativo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética
10.
Science ; 360(6389)2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29748257

RESUMEN

NAD+ (nicotinamide adenine dinucleotide in its oxidized state) is an essential molecule for a variety of physiological processes. It is synthesized in distinct subcellular compartments by three different synthases (NMNAT-1, -2, and -3). We found that compartmentalized NAD+ synthesis by NMNATs integrates glucose metabolism and adipogenic transcription during adipocyte differentiation. Adipogenic signaling rapidly induces cytoplasmic NMNAT-2, which competes with nuclear NMNAT-1 for the common substrate, nicotinamide mononucleotide, leading to a precipitous reduction in nuclear NAD+ levels. This inhibits the catalytic activity of poly[adenosine diphosphate (ADP)-ribose] polymerase-1 (PARP-1), a NAD+-dependent enzyme that represses adipogenic transcription by ADP-ribosylating the adipogenic transcription factor C/EBPß. Reversal of PARP-1-mediated repression by NMNAT-2-mediated nuclear NAD+ depletion in response to adipogenic signals drives adipogenesis. Thus, compartmentalized NAD+ synthesis functions as an integrator of cellular metabolism and signal-dependent transcriptional programs.


Asunto(s)
Adipocitos/citología , Adipogénesis/genética , NAD/biosíntesis , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Transcripción Genética , Adipocitos/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Glucosa/metabolismo , Humanos , Ratones , NAD/genética , Células 3T3 NIH , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Especificidad por Sustrato
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