RESUMEN
In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.
Asunto(s)
ADP-Ribosilación , Núcleo Celular/enzimología , Mitocondrias/enzimología , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Animales , Antimicina A/análogos & derivados , Antimicina A/farmacología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Transporte de Electrón/efectos de los fármacos , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Metacrilatos/farmacología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/enzimología , Oligomicinas/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Poli(ADP-Ribosa) Polimerasa-1/genética , Rotenona/farmacología , Tiazoles/farmacologíaRESUMEN
AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
Asunto(s)
Aterosclerosis/metabolismo , Aterosclerosis/patología , Médula Ósea/patología , Eliminación de Gen , Receptores Depuradores de Clase A/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Animales , Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/metabolismo , Trasplante de Médula Ósea , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Hematopoyesis , Homocigoto , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Modelos Biológicos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células RAW 264.7RESUMEN
While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.
RESUMEN
The clostridium-like ecto-ADP-ribosyltransferase ARTC1 is expressed in a highly restricted manner in skeletal muscle and heart tissue. Although ARTC1 is well studied, the identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level have been challenging and only a few ARTC1-ADP-ribosylated targets are known. Applying our recently developed mass spectrometry-based workflow to C2C12 myotubes and to skeletal muscle and heart tissues from wild-type mice, we identify hundreds of ARTC1-ADP-ribosylated proteins whose modifications are absent in the ADP-ribosylome of ARTC1-deficient mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport, and muscle function. Validation of hemopexin (HPX) as a ARTC1-target protein confirmed the functional importance of ARTC1-mediated extracellular arginine ADP-ribosylation at the systems level.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Hemopexina/metabolismo , Proteínas Musculares/genética , Debilidad Muscular/genética , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , ADP-Ribosilación , Animales , Arginina/metabolismo , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Ontología de Genes , Hemo/química , Hemo/metabolismo , Hemopexina/química , Hemopexina/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Anotación de Secuencia Molecular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/clasificación , Proteínas Musculares/metabolismo , Debilidad Muscular/metabolismo , Debilidad Muscular/patología , Músculo Esquelético/patología , Miocardio/patología , Unión Proteica , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Transducción de SeñalRESUMEN
Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521. In this protocol, we describe how to use this robust and versatile method for genome-wide and loci-specific localization of chromatin ADP-ribosylation. ADPr-ChAP enables bioinformatic comparisons of ADP-ribosylation with other chromatin modifications and is useful for understanding how ADP-ribosylation regulates biologically important cellular processes. ADPr-ChAP takes 1 week and requires standard skills in molecular biology and biochemistry. Although not covered in detail here, this technique can also be combined with conventional ChIP or DNA analysis to define the histone marks specifically associated with the ADP-ribosylated chromatin fractions and dissect the molecular mechanism and functional role of chromatin ADP-ribosylation.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Inmunoprecipitación de Cromatina/métodos , Cromatina/aislamiento & purificación , Cromatina/metabolismo , Adenosina Difosfato Ribosa/química , Animales , Línea Celular , Cromatina/química , HumanosRESUMEN
Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.