RESUMEN
Mitochondrial dysfunction is at the core of many diseases ranging from inherited metabolic diseases to common conditions that are associated with aging. Although associations between aging and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from Caenorhabditis elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-of-the-art platform for assessing respiration in cells, and we adapted the technique for applications involving C. elegans. Here we provide a detailed protocol to optimize and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes â¼2 d to complete, excluding the time spent culturing C. elegans, and it includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparation and execution of a run with the XF96 respirometer and (iv) postexperimental data analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the eight-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and the high throughput capacity due to the 96-well format.
Asunto(s)
Caenorhabditis elegans/citología , Mitocondrias/metabolismo , Animales , Bioquímica/instrumentación , Bioquímica/métodos , Caenorhabditis elegans/metabolismo , Respiración de la Célula , Diseño de Equipo , Consumo de OxígenoRESUMEN
To understand primary mitochondrial disease, we utilized a complex I-deficient Caenorhabditis elegans mutant, gas-1. These animals strongly upregulate the expression of gst-14 (encoding a glutathione S-transferase). Knockdown of gst-14 dramatically extends the lifespan of gas-1 and increases hydroxynonenal (HNE) modified mitochondrial proteins without improving complex I function. We observed no change in reactive oxygen species levels as measured by Mitosox staining, consistent with a potential role of GST-14 in HNE clearance. The upregulation of gst-14 in gas-1 animals is specific to the pharynx. These data suggest that an HNE-mediated response in the pharynx could be beneficial for lifespan extension in the context of complex I dysfunction in C. elegans. Thus, whereas HNE is typically considered damaging, our work is consistent with recent reports of its role in signaling, and that in this case, the signal is pro-longevity in a model of mitochondrial dysfunction.
Asunto(s)
Envejecimiento , Glutatión Transferasa/metabolismo , Mitocondrias/patología , Aldehídos/química , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Músculos/metabolismo , NADH Deshidrogenasa/metabolismo , Faringe/metabolismo , Faringe/fisiopatología , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Simple multicellular animal model systems are central to studying the complex mechanisms underlying a bewildering array of diseases involving dysfunctional mitochondria. Mutant nuclear- and mitochondrial-encoded subunits of the Caenorhabditis elegans mitochondrial respiratory chain (MRC) have been investigated, including GAS-1, NUO-1, NUO-6, MEV-1, SDHB-1, CLK-1, ISP-1, CTB-1, and ATP-2. These, as well as proteins that modify the MRC indirectly, have been studied on the molecular, cellular, and organismal levels through the variety of experimental approaches that are readily achievable in C. elegans. In C. elegans, MRC dysfunction can mimic signs and symptoms observed in human patients with primary mitochondrial disorders, such as neuromuscular deficits, developmental delay, altered anesthetic sensitivity, and increased lactate levels. Antioxidant dietary supplements, coenzyme Q substitutes, and flavin cofactors have been explored as potential therapeutic strategies. Furthermore, mutants with altered longevity have proved useful for probing the contributions of bioenergetics, reactive oxygen species, and stress responses to the process of aging. C. elegans will undoubtedly continue to provide a useful system in which to explore unanswered questions in mitochondrial biology and disease.
Asunto(s)
Caenorhabditis elegans/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Animales , Transporte de Electrón , Esperanza de Vida , Modelos BiológicosRESUMEN
How an organism ages is a question that has fascinated biologists, and the elderly, for centuries. One useful approach to understanding complex processes such as aging is to study genetic variation in model organisms such as the nematode, Caenorhabditis elegans. Classical mutant strains and RNAi screens have demonstrated that mitochondrial function is a major factor affecting longevity. Recent advances in the biochemical methods for studying mitochondrial functions have extended the usefulness of C. elegans for deciphering the molecular mechanisms by which mitochondria determine lifespan. Defects of all complexes in the mitochondrial respiratory chain have been described and have varied effects on lifespan. The phenotypes of these mutants indicate that the locality, production rate, and/or steady-state level of reactive oxygen species (ROS) is a defining lifespan-determining factor in these mutants. Mutants of enzymes involved in ROS scavenging have also been described, such as mitochondrial superoxide dismutases, and reveal a complex connection between ROS and lifespan. Energy balance, transcriptional signaling pathways, stress tolerance, and metabolic restructuring are also tied to ROS, and may also play roles in the mutants' altered lifespans. In this review, we discuss how findings with C. elegans genetics extend our understanding of the contributions of the mitochondrial respiratory chain and ROS to the process of aging.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Metabolismo Energético , Longevidad , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Edad , Envejecimiento/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Metabolismo Energético/genética , Genotipo , Longevidad/genética , Proteínas Mitocondriales/genética , Modelos Animales , FenotipoRESUMEN
Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to have critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.
Asunto(s)
Benzoatos/farmacología , Senescencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Melanoma/patología , Pirazoles/farmacología , Neoplasias Cutáneas/patología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Línea Celular Tumoral , Senescencia Celular/fisiología , Daño del ADN/fisiología , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/fisiología , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/metabolismo , Humanos , Melanoma/genética , Nitrobencenos , Pirazolonas , Neoplasias Cutáneas/genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell-based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET-based reporter, Histac, in live-cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, thus suggesting a role for p300/CBP in the acetylation of the histone H4 tail. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live-cell strategy for identifying and evaluating p300/CBP inhibitors.
Asunto(s)
Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.
Asunto(s)
Fosfoproteínas/análisis , Proteómica/métodos , Tirosina/metabolismo , Familia-src Quinasas/metabolismo , Animales , Western Blotting , Proteína Tirosina Quinasa CSK , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Adhesiones Focales , Factor 2 Liberador de Guanina Nucleótido/metabolismo , Humanos , Imidazoles/química , Espectrometría de Masas , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación , Fosfopéptidos/análisis , Fosforilación , Unión Proteica , Proto-Oncogenes Mas , Transducción de Señal , Especificidad por Sustrato , Tirosina/química , Tirosina/genética , Proteínas de Unión al GTP rap1/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.
Asunto(s)
Compuestos Aza/metabolismo , Colorantes Fluorescentes/metabolismo , Histona Desacetilasas/metabolismo , Histona Demetilasas/metabolismo , Lisina/metabolismo , Péptidos/metabolismo , Acetilación , Compuestos Aza/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Histona Desacetilasas/química , Histona Demetilasas/química , Lisina/análogos & derivados , Lisina/química , Metilación , Estructura Molecular , Péptidos/síntesis química , Péptidos/químicaRESUMEN
Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetyl-Lys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific antiacetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.
Asunto(s)
Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Acetilación , Alquilación , Animales , Aziridinas/química , Aziridinas/metabolismo , Sitios de Unión , Histonas/química , Histonas/metabolismo , Péptidos/química , Proteínas/química , Especificidad por SustratoRESUMEN
MAML1 is a transcriptional coregulator originally identified as a Notch coactivator. MAML1 is also reported to interact with other coregulator proteins, such as CDK8 and p300, to modulate the activity of Notch. We, and others, previously showed that MAML1 recruits p300 to Notch-regulated genes through direct interactions with the DNA-CSL-Notch complex and p300. MAML1 interacts with the C/H3 domain of p300, and the p300-MAML1 complex specifically acetylates lysines of histone H3 and H4 tails in chromatin in vitro. In this report, we show that MAML1 potentiates p300 autoacetylation and p300 transcriptional activation. MAML1 directly enhances p300 HAT activity, and this coincides with the translocation of MAML1, p300 and acetylated histones to nuclear bodies.
Asunto(s)
Transactivadores/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Histonas/metabolismo , Estructura Terciaria de Proteína , Eliminación de Secuencia , Transactivadores/química , Transactivadores/genética , Factores de Transcripción p300-CBP/químicaRESUMEN
Protein misfolding is monitored by a variety of cellular "quality control" systems. Endoplasmic reticulum (ER) quality control handles misfolded secretory and membrane proteins and is well characterized. However, less is known about the quality control of misfolded cytosolic proteins (CytoQC). To study CytoQC, we have employed a genetic system in Saccharomyces cerevisiae using a transplantable degron, CL1 (1). Attachment of CL1 to the cytosolic protein Ura3p destabilizes Ura3p, targeting it for rapid proteasomal degradation. We have performed a comprehensive analysis of Ura3p-CL1 degradation requirements. As shown previously, we observe that the ER-localized ubiquitin E2 (Ubc6p, Ubc7p, and Cue1p) and E3 (Doa10p) machinery involved in ER-associated degradation (ERAD) are also responsible for the degradation of the cytosolic substrate Ura3p-CL1. Importantly, we find that the cytosol/ER membrane-localized chaperones Ydj1p and Ssa1p, known to be necessary for the ERAD of membrane proteins with misfolded cytosolic domains, are also required for the ubiquitination and degradation of Ura3p-CL1. In addition, we show a role for the Cdc48p-Npl4p-Ufd1p complex in the degradation of Ura3p-CL1. When ubiquitination is blocked, a portion of Ura3p-CL1 is ER membrane-localized. Furthermore, access to the cytosolic face of the ER is required for the degradation of CL1 degron-containing proteins. The ER is distributed throughout the cytosol, and our data, together with previous studies, suggest that the cytosolic face of the ER membrane serves as a "platform" for the degradation of Ura3p-CL1, which may also be the case for other CytoQC substrates.