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A new mechanism for clearing protein damage from maturing oocytes has been described in a recent study by Bohnert and Kenyon (2017), who demonstrated that sperm-secreted hormones activate a vascular H+-ATPase pump that acidifies lysosomes and thus restores protein homeostasis.
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Caenorhabditis elegans , Proteostase , Animais , Transporte Biológico , Lisossomos , Masculino , Oócitos , ÓxidosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Label-free, non-contact imaging with mechanical contrast and optical sectioning is a substantial challenge in microscopy. Spontaneous Brillouin scattering microscopy meets this challenge, but encounters a trade-off between acquisition speed and the specificity for biomechanical constituents with overlapping Brillouin bands. Stimulated Brillouin scattering microscopy overcomes this trade-off and enables the cross-sectional imaging of live Caenorhabditis elegans at the organ and subcellular levels, with both elasticity and viscosity contrasts at high specificity and with practical recording times.
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Caenorhabditis elegans/fisiologia , Microscopia Confocal/métodos , Espalhamento de Radiação , Animais , Fenômenos Biomecânicos , Caenorhabditis elegans/microbiologia , Elasticidade , Escherichia coli , ViscosidadeRESUMO
BACKGROUND: Mitochondrial DNA (mtDNA) is present at high copy numbers in animal cells, and though characterized by a single haplotype in each individual due to maternal germline inheritance, deleterious mutations and intact mtDNA molecules frequently co-exist (heteroplasmy). A number of factors, such as replicative segregation, mitochondrial bottlenecks, and selection, may modulate the exitance of heteroplasmic mutations. Since such mutations may have pathological consequences, they likely survive and are inherited due to functional complementation via the intracellular mitochondrial network. Here, we hypothesized that compromised mitochondrial fusion would hamper such complementation, thereby affecting heteroplasmy inheritance. RESULTS: We assessed heteroplasmy levels in three Caenorhabditis elegans strains carrying different heteroplasmic mtDNA deletions (ΔmtDNA) in the background of mutant mitofusin (fzo-1). Animals displayed severe embryonic lethality and developmental delay. Strikingly, observed phenotypes were relieved during subsequent generations in association with complete loss of ΔmtDNA molecules. Moreover, deletion loss rates were negatively correlated with the size of mtDNA deletions, suggesting that mitochondrial fusion is essential and sensitive to the nature of the heteroplasmic mtDNA mutations. Introducing the ΔmtDNA into a fzo-1;pdr-1;+/ΔmtDNA (PARKIN ortholog) double mutant resulted in a skewed Mendelian progeny distribution, in contrast to the normal distribution in the fzo-1;+/ΔmtDNA mutant, and severely reduced brood size. Notably, the ΔmtDNA was lost across generations in association with improved phenotypes. CONCLUSIONS: Taken together, our findings show that when mitochondrial fusion is compromised, deleterious heteroplasmic mutations cannot evade natural selection while inherited through generations. Moreover, our findings underline the importance of cross-talk between mitochondrial fusion and mitophagy in modulating the inheritance of mtDNA heteroplasmy.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , DNA Mitocondrial/genética , GTP Fosfo-Hidrolases/genética , Padrões de Herança , Mitocôndrias/genética , Dinâmica Mitocondrial/genéticaRESUMO
Safeguarding the proteome is central to the health of the cell. In multi-cellular organisms, the composition of the proteome, and by extension, protein-folding requirements, varies between cells. In agreement, chaperone network composition differs between tissues. Here, we ask how chaperone expression is regulated in a cell type-specific manner and whether cellular differentiation affects chaperone expression. Our bioinformatics analyses show that the myogenic transcription factor HLH-1 (MyoD) can bind to the promoters of chaperone genes expressed or required for the folding of muscle proteins. To test this experimentally, we employed HLH-1 myogenic potential to genetically modulate cellular differentiation of Caenorhabditis elegans embryonic cells by ectopically expressing HLH-1 in all cells of the embryo and monitoring chaperone expression. We found that HLH-1-dependent myogenic conversion specifically induced the expression of putative HLH-1-regulated chaperones in differentiating muscle cells. Moreover, disrupting the putative HLH-1-binding sites on ubiquitously expressed daf-21(Hsp90) and muscle-enriched hsp-12.2(sHsp) promoters abolished their myogenic-dependent expression. Disrupting HLH-1 function in muscle cells reduced the expression of putative HLH-1-regulated chaperones and compromised muscle proteostasis during and after embryogenesis. In turn, we found that modulating the expression of muscle chaperones disrupted the folding and assembly of muscle proteins and thus, myogenesis. Moreover, muscle-specific over-expression of the DNAJB6 homolog DNJ-24, a limb-girdle muscular dystrophy-associated chaperone, disrupted the muscle chaperone network and exposed synthetic motility defects. We propose that cellular differentiation could establish a proteostasis network dedicated to the folding and maintenance of the muscle proteome. Such cell-specific proteostasis networks can explain the selective vulnerability that many diseases of protein misfolding exhibit even when the misfolded protein is ubiquitously expressed.
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Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Fatores de Regulação Miogênica/genética , Animais , Sítios de Ligação , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/biossíntese , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteínas Musculares , Fatores de Regulação Miogênica/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
Protein phosphorylation underlies cellular response pathways across eukaryotes and is governed by the opposing actions of phosphorylating kinases and de-phosphorylating phosphatases. While kinases and phosphatases have been extensively studied, their organization and the mechanisms by which they balance each other are not well understood. To address these questions we performed quantitative analyses of large-scale 'omics' datasets from yeast, fly, plant, mouse and human. We uncovered an asymmetric balance of a previously-hidden scale: Each organism contained many different kinase genes, and these were balanced by a small set of highly abundant phosphatase proteins. Kinases were much more responsive to perturbations at the gene and protein levels. In addition, kinases had diverse scales of phenotypic impact when manipulated. Phosphatases, in contrast, were stable, highly robust and flatly organized, with rather uniform impact downstream. We validated aspects of this organization experimentally in nematode, and supported additional aspects by theoretic analysis of the dynamics of protein phosphorylation. Our analyses explain the empirical bias in the protein phosphorylation field toward characterization and therapeutic targeting of kinases at the expense of phosphatases. We show quantitatively and broadly that this is not only a historical bias, but stems from wide-ranging differences in their organization and impact. The asymmetric balance between these opposing regulators of protein phosphorylation is also common to opposing regulators of two other post-translational modification systems, suggesting its fundamental value.
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Evolução Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/genética , Fosfotransferases/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ativação Enzimática/genética , Variação Genética/genética , Camundongos , Monoéster Fosfórico Hidrolases/classificação , Fosforilação , Fosfotransferases/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , LevedurasRESUMO
Emerging antibiotic resistance among human pathogens has galvanized efforts to find alternative routes to combat bacterial virulence. One new approach entails interfering with the ability of bacteria to coordinate population-wide gene expression, or quorum sensing (QS), thus inhibiting the production of virulence factors and biofilm formation. We have recently developed such a strategy by targeting LasR, the master regulator of QS in the opportunistic human pathogen Pseudomonas aeruginosa, through the rational design of covalent inhibitors closely based on the core structure of the native ligand. We now report several groups of new inhibitors, one of which, fluoro-substituted ITC-12, displayed complete covalent modification of LasR, as well as effective QS inhibition in vitro and promising in vivo results. In addition to their potential clinical relevance, this series of synthetic QS modulators can be used as a tool to further unravel the complicated QS regulation in P.â aeruginosa.
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Antibacterianos/farmacologia , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/efeitos dos fármacos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Antibacterianos/química , Proteínas de Bactérias/agonistas , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biofilmes/efeitos dos fármacos , Domínio Catalítico , Dicroísmo Circular , Isotiocianatos/química , Isotiocianatos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Estresse Oxidativo/efeitos dos fármacos , Transativadores/agonistas , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fatores de Virulência/metabolismoRESUMO
Protein folding and clearance networks sense and respond to misfolded and aggregation-prone proteins by activating cytoprotective cell stress responses that safeguard the proteome against damage, maintain the health of the cell, and enhance lifespan. Surprisingly, cellular proteostasis undergoes a sudden and widespread failure early in Caenorhabditis elegans adulthood, with marked consequences on proteostasis functions later in life. These changes in the regulation of quality control systems, such as chaperones, the ubiquitin proteasome system and cellular stress responses, are controlled cell-nonautonomously by the proliferation of germline stem cells. Here, we review recent studies examining changes in proteostasis upon transition to adulthood and how proteostasis is modulated by reproduction onset, focusing on C. elegans. Based on these and our own findings, we propose that the regulation of quality control systems is actively remodeled at the point of transition between development and adulthood to influence the subsequent course of aging.
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Bacterial communication, Quorum Sensing (QS), is a target against virulence and prevention of antibiotic-resistant infections. 16 derivatives of Piperlongumine (PL), an amide alkaloid from Piper longum L., were screened for QS inhibition. PL-18 had the best QSI activity. PL-18 inhibited the lasR-lasI, rhlR-rhlI, and pqs QS systems of Pseudomonas aeruginosa. PL-18 inhibited pyocyanin and rhamnolipids that are QS-controlled virulence elements. Iron is an essential element for pathogenicity, biofilm formation and resilience in harsh environments, its uptake was inhibited by PL-18. Pl-18 significantly reduced the biofilm biovolume including in established biofilms. PL-18-coated silicon tubes significantly inhibited biofilm formation. The transcriptome study of treated P. aeruginosa showed that PL-18 indeed reduced the expression of QS and iron homeostasis related genes, and up regulated sulfur metabolism related genes. Altogether, PL-18 inhibits QS, virulence, iron uptake, and biofilm formation. Thus, PL-18 should be further developed against bacterial infection, antibiotic resistance, and biofilm formation.
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Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the pre-initiation complex at the 5' end of mRNAs driving translation initiation. The genome of Leishmania encodes a large repertoire of cap-binding complexes that fulfill a variety of functions possibly involved in survival along the life cycle. However, most of these complexes function in the promastigote life form that resides in the sand fly vector and decrease their activity in amastigotes, the mammalian life form. Here we examined the possibility that LeishIF3d drives translation in Leishmania using alternative pathways. We describe a non-canonical cap-binding activity of LeishIF3d and examine its potential role in driving translation. LeishIF3d is required for translation, as reducing its expression by a hemizygous deletion reduces the translation activity of the LeishIF3d(+/-) mutant cells. Proteomic analysis of the mutant cells highlights the reduced expression of flagellar and cytoskeletal proteins, as reflected in the morphological changes observed in the mutant cells. Targeted mutations in two predicted alpha helices diminish the cap-binding activity of LeishIF3d. Overall, LeishIF3d could serve as a driving force for alternative translation pathways, although it does not seem to offer an alternative pathway for translation in amastigotes.
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The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.
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Chaperonas Moleculares , Estresse Fisiológico , Humanos , Proteínas de Choque Térmico HSP70 , Chaperonas Moleculares/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.
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Proteínas de Choque Térmico Pequenas , Proteínas de Choque Térmico Pequenas/metabolismo , ProteínasRESUMO
Protein damage contributes prominently to cellular aging. To address whether this occurs at a specific period during aging or accumulates gradually, we monitored the biochemical, cellular, and physiological properties of folding sensors expressed in different tissues of C. elegans. We observed the age-dependent misfolding and loss of function of diverse proteins harboring temperature-sensitive missense mutations in all somatic tissues at the permissive condition. This widespread failure in proteostasis occurs rapidly at an early stage of adulthood, and coincides with a severely reduced activation of the cytoprotective heat shock response and the unfolded protein response. Enhancing stress responsive factors HSF-1 or DAF-16 suppresses misfolding of these metastable folding sensors and restores the ability of the cell to maintain a functional proteome. This suggests that a compromise in the regulation of proteostatic stress responses occurs early in adulthood and tips the balance between the load of damaged proteins and the proteostasis machinery. We propose that the collapse of proteostasis represents an early molecular event of aging that amplifies protein damage in age-associated diseases of protein conformation.
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Envelhecimento , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Amplificação de Genes , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Conformação Proteica , Dobramento de ProteínaRESUMO
Chaperone expression is developmentally regulated, establishing tissue-specific networks. However, the molecular basis underlying this specificity is mainly unknown. Recent evidence suggests that chaperone network rewiring is mediated, in part, by differentiation transcription factors to fit the proteome folding demands, with implications for the tissue-specific manifestation of protein misfolding diseases.
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Chaperonas Moleculares , Deficiências na Proteostase , Diferenciação Celular , Humanos , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteoma/metabolismo , Deficiências na Proteostase/metabolismo , Fatores de TranscriçãoRESUMO
Muscle proteostasis is shaped by the myogenic transcription factor MyoD which regulates the expression of chaperones during muscle differentiation. Whether MyoD can also modulate chaperone expression in terminally differentiated muscle cells remains open. Here we utilized a temperature-sensitive (ts) conditional knockdown nonsense mutation in MyoD ortholog in C. elegans, HLH-1, to ask whether MyoD plays a role in maintaining muscle proteostasis post myogenesis. We showed that hlh-1 is expressed during larval development and that hlh-1 knockdown at the first, second, or third larval stages resulted in severe defects in motility and muscle organization. Motility defects and myofilament organization were rescued when the clearance of hlh-1(ts) mRNA was inhibited, and hlh-1 mRNA levels were restored. Moreover, hlh-1 knockdown modulated the expression of chaperones with putative HLH-1 binding sites in their promoters, supporting HLH-1 role in muscle maintenance during larval development. Finally, mild disruption of hlh-1 expression during development resulted in earlier dysregulation of muscle maintenance and function during adulthood. We propose that the differentiation transcription factor, HLH-1, contributes to muscle maintenance and regulates cell-specific chaperone expression post differentiation. HLH-1 may thus impact muscle proteostasis and potentially the onset and manifestation of sarcopenia.
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The quality control machinery regulates the cellular proteome to ensure proper protein homeostasis (proteostasis). In Caenorhabditis elegans, quality control networks are downregulated cell-nonautonomously by the gonadal longevity pathway or metabolic signaling at the onset of reproduction. However, how signals are mediated between the gonad and the somatic tissues is not known. Gonadotropin-releasing hormone (GnRH)-like signaling functions in the interplay between development and reproduction and have conserved roles in regulating reproduction, metabolism, and stress. We, therefore, asked whether GnRH-like signaling is involved in proteostasis collapse at the onset of reproduction. Here, we examine whether C. elegans orthologues of GnRH receptors modulate heat shock survival. We find that gnrr-2 is required for proteostasis remodeling in different somatic tissues during the transition to adulthood. We show that gnrr-2 likely functions in neurons downstream of the gonad in the gonadal-longevity pathway and modulate the somatic regulation of transcription factors HSF-1, DAF-16, and PQM-1. In parallel, gnrr-2 modulates egg-laying rates, vitellogenin production, and thus reproductive capacity. Taken together, our data suggest that gnrr-2 plays a GnRH-associated role, mediating the cross-talk between the reproduction system and the soma in the decision to commit to reproduction.
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The sensitivity of the protein-folding environment to chaperone disruption can be highly tissue-specific. Yet, the organization of the chaperone system across physiological human tissues has received little attention. Through computational analyses of large-scale tissue transcriptomes, we unveil that the chaperone system is composed of core elements that are uniformly expressed across tissues, and variable elements that are differentially expressed to fit with tissue-specific requirements. We demonstrate via a proteomic analysis that the muscle-specific signature is functional and conserved. Core chaperones are significantly more abundant across tissues and more important for cell survival than variable chaperones. Together with variable chaperones, they form tissue-specific functional networks. Analysis of human organ development and aging brain transcriptomes reveals that these functional networks are established in development and decline with age. In this work, we expand the known functional organization of de novo versus stress-inducible eukaryotic chaperones into a layered core-variable architecture in multi-cellular organisms.
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Chaperonas Moleculares/metabolismo , Especificidade de Órgãos , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Linhagem Celular , Sequência Conservada , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Camundongos , Chaperonas Moleculares/genética , Fases de Leitura Aberta/genética , Especificidade de Órgãos/genéticaRESUMO
One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.
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Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico/metabolismo , Desenvolvimento Muscular/imunologia , Músculo Esquelético/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular , Células HeLa , Humanos , Músculo Esquelético/citologia , Transfecção , Receptor de Lamina BRESUMO
Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.
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Caenorhabditis elegans/metabolismo , Programas de Rastreamento , Chaperonas Moleculares/metabolismo , Especificidade de Órgãos , Animais , Bioensaio , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Embrião não Mamífero/metabolismo , Epistasia Genética , Mitocôndrias/metabolismo , Óvulo/metabolismo , Fenótipo , Ligação Proteica , Dobramento de Proteína , Proteoma/metabolismo , Interferência de RNA , Reprodutibilidade dos TestesRESUMO
Protein homeostasis is remodeled early in Caenorhabditis elegans adulthood, resulting in a sharp decline in folding capacity and reduced ability to cope with chronic and acute stress. Endocrine signals from the reproductive system can ameliorate this proteostatic collapse and reshape the quality control network. Given that environmental conditions, such as food availability, impact reproductive success, we asked whether conditions of dietary restriction (DR) can also reverse the decline in quality control function at the transition to adulthood, and if so, whether gonadal signaling and dietary signaling remodel the quality control network in a similar or different manner. For this, we employed the eat-2 genetic model and bacterial deprivation protocol. We found that animals under DR maintained heat shock response activation and high protein folding capacity during adulthood. However, while gonadal signaling required DAF-16, DR-associated rescue of quality control functions required the antagonistic transcription factor, PQM-1. Bioinformatic analyses supported a role for DAF-16 in acute stress responses and a role for PQM-1 in cellular maintenance and chronic stress. Comparing the stress activation and folding capacities of dietary- and gonadal-signaling mutant animals confirmed this prediction and demonstrated that each differentially impacts cellular quality control capabilities. These data suggest that the functional mode of cellular quality control networks can be differentially remodeled, affecting an organism's ability to respond to acute and chronic stresses during adulthood.