RESUMO
Though the effect of the recently identified mitochondrial NAD+ transporter SLC25A51 on glucose metabolism has been described, its contribution to other NAD+-dependent processes throughout the cell such as ADP-ribosylation remains elusive. Here, we report that absence of SLC25A51 leads to increased NAD+ concentration not only in the cytoplasm and but also in the nucleus. The increase is not associated with upregulation of the salvage pathway, implying an accumulation of constitutively synthesized NAD+ in the cytoplasm and nucleus. This results in an increase of PARP1-mediated nuclear ADP-ribosylation, as well as faster repair of DNA lesions induced by different single-strand DNA damaging agents. Lastly, absence of SLC25A51 reduces both MMS/Olaparib induced PARP1 chromatin retention and the sensitivity of different breast cancer cells to PARP1 inhibition. Together these results provide evidence that SLC25A51 might be a novel target to improve PARP1 inhibitor based therapies by changing subcellular NAD+ redistribution.
Assuntos
NAD , Cromatina , Reparo do DNA , Mitocôndrias/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , HumanosRESUMO
ADP-ribosylation is a posttranslational protein modification, involved in various cellular processes, ranging from DNA-damage repair to apoptosis. While its function has been studied amply with respect to genotoxic stress-associated nuclear ADP-ribosylation, the functional relevance of mitochondrial ADP-ribosylation remains so far poorly studied. This is mainly attributed to the absence of powerful techniques able to detect the modification. However, the usage of recently developed anti-ADP-ribose-specific antibodies allows now to investigate mitochondrial ADP-ribosylation under physiological and pathophysiological conditions. In the below method, we describe in detail how to efficiently detect and quantify mitochondrial ADP-ribosylation via immunofluorescence.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Imunofluorescência/métodos , Mitocôndrias/metabolismo , ADP-Ribosilação , Animais , Células Cultivadas , HumanosRESUMO
Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD+)-dependent post-translational modification that is found on proteins as well as on nucleic acids. While ARTD1/PARP1-mediated poly-ADP-ribosylation has extensively been studied in the past 60 years, comparably little is known about the physiological function of mono-ADP-ribosylation and the enzymes involved in its turnover. Promising technological advances have enabled the development of innovative tools to detect NAD+ and NAD+/NADH (H for hydrogen) ratios as well as ADP-ribosylation. These tools have significantly enhanced our current understanding of how intracellular NAD dynamics contribute to the regulation of ADP-ribosylation as well as to how mono-ADP-ribosylation integrates into various cellular processes. Here, we discuss the recent technological advances, as well as associated new biological findings and concepts.
Assuntos
Adenosina Difosfato Ribose/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , ADP-Ribosilação/fisiologia , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
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.
Assuntos
ADP-Ribosilação , Núcleo Celular/enzimologia , Mitocôndrias/enzimologia , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Metacrilatos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/enzimologia , Oligomicinas/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Poli(ADP-Ribose) Polimerase-1/genética , Rotenona/farmacologia , Tiazóis/farmacologiaRESUMO
Cells constantly adapt their metabolic pathways to meet their energy needs and respond to nutrient availability. During the last two decades, it has become increasingly clear that NAD+, a coenzyme in redox reactions, also mediates several ubiquitous cell signaling processes. Protein ADP-ribosylation is a post-translational modification that uses NAD+ as a substrate and is best known as part of the genotoxic stress response. However, there is increasing evidence that NAD+-dependent ADP-ribosylation regulates other cellular processes, including metabolic pathways. In this review, we will describe the compartmentalized regulation of NAD+ biosynthesis, consumption, and regeneration with a particular focus on the role of ADP-ribosylation in the regulation of glucose metabolism in different cellular compartments.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Glucose/metabolismo , NAD/metabolismo , Organelas/metabolismo , ADP-Ribosilação , Animais , Metabolismo dos Carboidratos/fisiologia , Linhagem Celular , Dano ao DNA/fisiologia , Humanos , Redes e Vias Metabólicas/fisiologia , CamundongosRESUMO
Mice deficient for ADP-ribosyltransferase diphteria toxin-like 1 (ARTD1) are protected against microbially induced inflammation. To address the contribution of ARTD1 to inflammation specifically in myeloid cells, we generated an Artd1ΔMyel mouse strain with conditional ARTD1 deficiency in myeloid lineages and examined the strain in three disease models. We found that ARTD1, but not its enzymatic activity, enhanced the transcriptional activation of distinct LPS-induced genes that included IL-12, TNF-α, and IL-6 in primary bone marrow-derived macrophages and LPS-induced IL-12/18-IFN-γ signaling in Artd1ΔMyel mice. The loss of Artd1 in myeloid cells also reduced the TH1 response to Helicobacter pylori and impaired immune control of the bacteria. Furthermore, Artd1ΔMyel mice failed to control tumor growth in a s.c. MC-38 model of colon cancer, which could be attributed to reduced TH1 and CD8 responses. Together, these data provide strong evidence for a cell-intrinsic role of ARTD1 in myeloid cells that is independent of its enzymatic activity and promotes type I immunity by promoting IL-12/18 expression.
Assuntos
Infecções por Helicobacter/imunologia , Modelos Imunológicos , Células Mieloides/imunologia , Neoplasias/imunologia , Poli(ADP-Ribose) Polimerase-1/imunologia , Sepse/imunologia , Animais , Células Cultivadas , Biologia Computacional , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-18/genética , Interleucina-18/imunologia , CamundongosRESUMO
OBJECTIVE: The advent of ligand-based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time-consuming. Here, we present a methodology that provides a fast and cost-efficient alternative for candidate target verification on living cells. RESULTS: In the described methodology a ligand of interest (e.g. transferrin, epidermal growth factor or insulin) was conjugated to a linker (TriCEPS) that carries a biotin. To confirm ligand/receptor interactions, the ligand-TriCEPS conjugates were first added onto living cells and cells were subsequently labeled with a streptavidin-fluorophore and analyzed by flow cytometry (thus referred as Flow-TriCEPS). Flow-TriCEPS was also used to validate identified receptor candidates when combined with a siRNA knock down approach (i.e. reduction of expression levels). This approach is versatile as it can be applied for different classes of ligands (proteins, peptides, antibodies) and different cell lines. Moreover, the method is time-efficient since it takes advantage of the large variety of commercially available (and certified) siRNAs.
Assuntos
Biotina/análogos & derivados , Receptores ErbB/metabolismo , Citometria de Fluxo/métodos , Hidrazinas/metabolismo , Succinimidas/metabolismo , Biotina/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Insulina/metabolismo , Ligantes , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos TestesRESUMO
Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes.
Assuntos
Adenosina Difosfato Ribose/química , Bioensaio/métodos , Espectrometria de Massas/métodos , N-Glicosil Hidrolases/isolamento & purificação , Humanos , N-Glicosil Hidrolases/química , Processamento de Proteína Pós-TraducionalRESUMO
ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.