RESUMO
Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPR(mt)), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPR(mt) signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPR(mt) induction, while gain of function is sufficient to extend lifespan in a UPR(mt)-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPR(mt) signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Animais , Caenorhabditis elegans/genética , Longevidade , Camundongos , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
Nicotinamide adenine dinucleotide (NAD+) is a co-substrate for several enzymes, including the sirtuin family of NAD+-dependent protein deacylases. Beneficial effects of increased NAD+ levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-ß-carboxymuconate-ε-semialdehyde in the de novo NAD+ synthesis pathway, controls cellular NAD+ levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD+ synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD+ levels, sirtuin activity and mitochondrial homeostasis in kidney and liver.
Assuntos
Carboxiliases/metabolismo , Sequência Conservada , Evolução Molecular , Saúde , Mitocôndrias/fisiologia , NAD/biossíntese , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Carboxiliases/antagonistas & inibidores , Carboxiliases/química , Carboxiliases/deficiência , Linhagem Celular , Colina , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Masculino , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Ratos , Sirtuínas/metabolismoRESUMO
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.
Assuntos
Crinivirus/metabolismo , Proteína Catiônica de Eosinófilo/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Iridovirus/metabolismo , Interferência de RNA/fisiologia , Proteínas Virais/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/virologia , Immunoblotting , Mutagênese Sítio-Dirigida , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , RNA de Cadeia Dupla , RNA Interferente Pequeno/biossíntese , Nicotiana/virologia , TransfecçãoRESUMO
Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Reparo do DNA , Proteoma , Transcriptoma , Proteína de Xeroderma Pigmentoso Grupo A/genética , Animais , Antioxidantes/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , DNA Glicosilases/genética , Endonucleases/genética , Mutação , Purinas/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMO
SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1-mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elongação Traducional da Cadeia Peptídica , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Complexo de Endopeptidases do Proteassoma/genética , Fatores de Transcrição/genética , Ubiquitina/genéticaRESUMO
Milk-derived peptides and milk fat globule membrane (MFGM) have gained interest as health-promoting food ingredients. However, the mechanisms by which these nutraceuticals modulate the function of biological systems often remain unclear. We utilized Caenorhabditis elegans to elucidate how MFGM-containing protein powder (MProPow), previously used in a clinical trial, affect the physiology of this model organism. Our results demonstrate that MProPow does not affect lifespan but promotes the fitness of the animals. Surprisingly, gene expression analysis revealed that MProPow decreases the expression of genes functioning on innate immunity, which also translates into reduced survival on pathogenic bacteria. One of the innate immunity-associated genes showing reduced expression upon MProPow supplementation is cpr-3, the homolog of human cathepsin B. Interestingly, knockdown of cpr-3 enhances fitness, but not in MProPow-treated animals, suggesting that MProPow contributes to fitness by downregulating the expression of this gene. In summary, this research highlights the value of C. elegans in testing the biological activity of food supplements and nutraceuticals. Furthermore, this study should encourage investigations into whether milk-derived peptides and MFGM mediate their beneficial effects through the modulation of cathepsin B expression in humans.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Suplementos Nutricionais , Glicolipídeos , Glicoproteínas , Gotículas Lipídicas , Animais , Caenorhabditis elegans/efeitos dos fármacos , Glicolipídeos/farmacologia , Glicoproteínas/farmacologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Imunidade Inata/efeitos dos fármacos , Catepsina B/metabolismo , Pós , Proteínas do Leite/farmacologia , Longevidade/efeitos dos fármacosRESUMO
Folate receptor (FR) alpha (FOLR1) and beta (FOLR2) are membrane-anchored folate transporters that are expressed at low levels in normal tissues, while their expression is strongly increased in several cancers. Intriguingly, although the function of these receptors in, for example, development and cancer has been studied intensively, their role in aging is still unknown. To address this, we utilized Caenorhabditis elegans, in which FOLR-1 is the sole ortholog of folate receptors. We found that the loss of FOLR-1 does not affect reproduction, physical condition, proteostasis or lifespan, indicating that it is not required for folate transport to maintain health. Interestingly, we found that FOLR-1 is detectably expressed only in uterine-vulval cells, and that the histone-binding protein LIN-53 inhibits its expression in other tissues. Furthermore, whereas knockdown of lin-53 is known to shorten lifespan, we found that the loss of FOLR-1 partially rescues this phenotype, suggesting that elevated folr-1 expression is detrimental for health. Indeed, our data demonstrate that overexpression of folr-1 is toxic, and that this phenotype is dependent on diet. Altogether, this work could serve as a basis for further studies to elucidate the organismal effects of abnormal FR expression in diseases such as cancer.
Assuntos
Proteínas de Caenorhabditis elegans , Receptor 2 de Folato , Neoplasias , Animais , Feminino , Humanos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Receptor 1 de Folato/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido Fólico/metabolismo , Dieta , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Receptor 2 de Folato/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Histone H3/H4 chaperone anti-silencing function 1 (ASF1) is a conserved factor mediating nucleosomal assembly and disassembly, playing crucial roles in processes such as replication, transcription, and DNA repair. Nevertheless, its involvement in aging has remained unclear. Here, we utilized the model organism Caenorhabditis elegans to demonstrate that the loss of UNC-85, the homolog of ASF1, leads to a shortened lifespan in a multicellular organism. Furthermore, we show that UNC-85 is required for epigenome-mediated longevity, as knockdown of the histone H3 lysine K4 methyltransferase ash-2 does not extend the lifespan of unc-85 mutants. In this context, we found that the longevity-promoting ash-2 RNA interference enhances UNC-85 activity by increasing its nuclear localization. Finally, our data indicate that the loss of UNC-85 increases the activity of one-carbon metabolism, and that downregulation of the one-carbon metabolism component dao-3/MTHFD2 partially rescues the short lifespan of unc-85 mutants. Together, these findings reveal UNC-85/ASF1 as a modulator of the central metabolic pathway and a factor regulating a pro-longevity response, thus shedding light on a mechanism of how nucleosomal maintenance associates with aging.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Longevidade , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Animais , Longevidade/genética , Carbono/metabolismo , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Histonas/metabolismo , Interferência de RNA , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Epigênese GenéticaRESUMO
The ubiquitin-proteasome system (UPS) orchestrates many cellular and tissue-specific processes by degrading damaged and key regulatory proteins. To enable investigation of UPS activity in different cell types in a living animal, we developed a photoconvertible fluorescent UPS reporter system for live imaging and quantification of protein degradation in Caenorhabditis elegans. Our reporter consists of the photoconvertible fluorescent protein Dendra2 targeted for proteasomal degradation by fusion to the UbG76V mutant form of ubiquitin. In contrast to previous reporters, this system permits quantification of UPS activity independently of protein synthesis. Our reporter revealed that UPS-mediated protein degradation varies in a cell type-specific and age-dependent manner in C. elegans.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas Luminescentes/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitina/metabolismo , Envelhecimento/metabolismo , Animais , Proteínas de Caenorhabditis elegans/fisiologia , Dopamina/fisiologia , Microscopia de Fluorescência , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Muscleblind-like splicing regulators (MBNLs) are RNA-binding factors that have an important role in developmental processes. Dysfunction of these factors is a key contributor of different neuromuscular degenerative disorders, including Myotonic Dystrophy type 1 (DM1). Since DM1 is a multisystemic disease characterized by symptoms resembling accelerated aging, we asked which cellular processes do MBNLs regulate that make them necessary for normal lifespan. By utilizing the model organism Caenorhabditis elegans, we found that loss of MBL-1 (the sole ortholog of mammalian MBNLs), which is known to be required for normal lifespan, shortens lifespan by decreasing the activity of p38 MAPK/PMK-1 as well as the function of transcription factors ATF-7 and SKN-1. Furthermore, we show that mitochondrial stress caused by the knockdown of mitochondrial electron transport chain components promotes the longevity of mbl-1 mutants in a partially PMK-1-dependent manner. Together, the data establish a mechanism of how DM1-associated loss of muscleblind affects lifespan. Furthermore, this study suggests that mitochondrial stress could alleviate symptoms caused by the dysfunction of muscleblind splicing factor, creating a potential approach to investigate for therapy.
Assuntos
Fatores Ativadores da Transcrição/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Longevidade/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Variation in ambient growth temperature can cause changes in normal animal physiology and cellular functions such as control of protein homeostasis. A key mechanism for maintaining proteostasis is the selective degradation of polyubiquitinated proteins, mediated by the ubiquitin-proteasome system (UPS). It is still largely unsolved how temperature changes affect the UPS at the organismal level. Caenorhabditis elegans nematodes are normally bred at 20 °C, but for some experimental conditions, 25 °C is often used. We studied the effect of 25 °C on C. elegans UPS by measuring proteasome activity and polyubiquitinated proteins both in vitro in whole animal lysates and in vivo in tissue-specific transgenic reporter strains. Our results show that an ambient temperature shift from 20 to 25 °C increases the UPS activity in the intestine, but not in the body wall muscle tissue, where a concomitant accumulation of polyubiquitinated proteins occurs. These changes in the UPS activity and levels of polyubiquitinated proteins were not detectable in whole animal lysates. The exposure of transgenic animals to 25 °C also induced ER stress reporter fluorescence, but not the fluorescence of a heat shock responsive reporter, albeit detection of a mild induction in hsp-16.2 mRNA levels. In conclusion, C. elegans exhibits tissue-specific responses of the UPS as an organismal strategy to cope with a rise in ambient temperature.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Temperatura , Ubiquitina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Intestinos , Músculos/metabolismo , Especificidade de Órgãos , Proteínas Ubiquitinadas/metabolismoRESUMO
Myotonic dystrophy type 1 is an autosomal-dominant inherited disorder caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. The RNAs bearing these expanded repeats have a range of toxic effects. Here we provide evidence from a Caenorhabditis elegans myotonic dystrophy type 1 model that the RNA interference (RNAi) machinery plays a key role in causing RNA toxicity and disease phenotypes. We show that the expanded repeats systematically affect a range of endogenous genes bearing short non-pathogenic repeats and that this mechanism is dependent on the small RNA pathway. Conversely, by perturbating the RNA interference machinery, we reversed the RNA toxicity effect and reduced the disease pathogenesis. Our results unveil a role for RNA repeats as templates (based on sequence homology) for moderate but constant gene silencing. Such a silencing effect affects the cell steady state over time, with diverse impacts depending on tissue, developmental stage, and the type of repeat. Importantly, such a mechanism may be common among repeats and similar in human cells with different expanded repeat diseases.
Assuntos
Envelhecimento/genética , Caenorhabditis elegans/genética , Distrofia Miotônica/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Repetições de Trinucleotídeos , Regiões 3' não Traduzidas , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Humanos , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Ligação Proteica , RNA de Cadeia Dupla/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismoRESUMO
Through epigenetic mechanisms cells integrate environmental stimuli to fine-tune gene expression levels. Mitochondrial function is essential to provide the intermediate metabolites necessary to generate and modify epigenetic marks in the nucleus, which in turn can regulate the expression of mitochondrial proteins. In this review we summarize the function of mitochondria in the regulation of epigenetic mechanisms as a new aspect of mitonuclear communication. We focus in particular on the most common epigenetic modifications - histone acetylation and histone and DNA methylation. We also discuss the emerging field of mitochondrial DNA (mtDNA) methylation, whose physiological role remains unknown. Finally, we describe the essential role of some histone modifications in regulating the mitochondrial unfolded protein response (UPRmt) and the mitochondrial stress-dependent lifespan extension.
Assuntos
Epigênese Genética , Homeostase , Mitocôndrias/metabolismo , Estresse Fisiológico/genética , Animais , Cromatina/metabolismo , Metilação de DNA , DNA Mitocondrial/genética , HumanosRESUMO
Age-associated changes in chromatin structure have a major impact on organismal longevity. Despite being a central part of the ageing process, the organismal responses to the changes in chromatin organization remain unclear. Here we show that moderate disturbance of histone balance during C. elegans development alters histone levels and triggers a stress response associated with increased expression of cytosolic small heat-shock proteins. This stress response is dependent on the transcription factor, HSF-1, and the chromatin remodeling factor, ISW-1. In addition, we show that mitochondrial stress during developmental stages also modulates histone levels, thereby activating a cytosolic stress response similar to that caused by changes in histone balance. These data indicate that histone and mitochondrial perturbations are both monitored through chromatin remodeling and involve the activation of a cytosolic response that affects organismal longevity. HSF-1 and ISW-1 hence emerge as a central mediator of this multi-compartment proteostatic response regulating longevity.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Montagem e Desmontagem da Cromatina/fisiologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico Pequenas/metabolismo , Longevidade/fisiologia , Pressão Osmótica , Estresse Psicológico , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Humanos , Proteostase/genética , Proteostase/fisiologiaRESUMO
UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Nucléolo Celular/metabolismo , Humanos , Nucleofosmina , Inibidores de Proteassoma/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/efeitos da radiação , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Estresse Fisiológico , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Ubiquitina/metabolismo , Raios Ultravioleta/efeitos adversosRESUMO
The proteasome plays an important role in proteostasis by carrying out controlled protein degradation in the cell. Impairments in proteasome function are associated with severe and often age-related diseases. Here, we have characterized a molecular mechanism linking insulin/IGF-1 signaling (IIS) to proteasome activity. We show that decreased IIS, which promotes proteostasis and longevity, increases proteasome activity through the FOXO transcription factor DAF-16 in C. elegans. Furthermore, we reveal that DAF-16 represses expression of the proteasome-associated deubiquitinating enzyme ubh-4, which we suggest functions as a tissue-specific proteasome inhibitor. Finally, we demonstrate that proteasome activation through downregulation of the ubh-4 human ortholog uchl5 increases degradation of proteotoxic proteins in mammalian cells. In conclusion, we have established a mechanism by which the evolutionarily conserved IIS contributes to the regulation of proteasome activity in a multicellular organism.