RESUMO
Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.
Assuntos
Chaperonas Moleculares/genética , Técnicas de Sonda Molecular , Proteoma/genética , Deficiências na Proteostase/genética , Proteostase/genética , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Meia-Vida , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Chaperonas Moleculares/metabolismo , Agregados Proteicos , Engenharia de Proteínas/métodos , Dobramento de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteoma/química , Proteoma/metabolismo , Proteostase/efeitos dos fármacos , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Cellular functioning requires the orchestration of thousands of molecular interactions in time and space. Yet most molecules in a cell move by diffusion, which is sensitive to external factors like temperature. How cells sustain complex, diffusion-based systems across wide temperature ranges is unknown. Here, we uncover a mechanism by which budding yeast modulate viscosity in response to temperature and energy availability. This "viscoadaptation" uses regulated synthesis of glycogen and trehalose to vary the viscosity of the cytosol. Viscoadaptation functions as a stress response and a homeostatic mechanism, allowing cells to maintain invariant diffusion across a 20°C temperature range. Perturbations to viscoadaptation affect solubility and phase separation, suggesting that viscoadaptation may have implications for multiple biophysical processes in the cell. Conditions that lower ATP trigger viscoadaptation, linking energy availability to rate regulation of diffusion-controlled processes. Viscoadaptation reveals viscosity to be a tunable property for regulating diffusion-controlled processes in a changing environment.
Assuntos
Metabolismo Energético , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Temperatura , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Difusão , Glicogênio/metabolismo , Homeostase , Modelos Biológicos , Solubilidade , Trealose , ViscosidadeRESUMO
Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/fisiologia , Expressão Gênica/genética , Genes Essenciais/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Stress granules (SGs) are transient ribonucleoprotein (RNP) aggregates that form during cellular stress and are increasingly implicated in human neurodegeneration. To study the proteome and compositional diversity of SGs in different cell types and in the context of neurodegeneration-linked mutations, we used ascorbate peroxidase (APEX) proximity labeling, mass spectrometry, and immunofluorescence to identify â¼150 previously unknown human SG components. A highly integrated, pre-existing SG protein interaction network in unstressed cells facilitates rapid coalescence into larger SGs. Approximately 20% of SG diversity is stress or cell-type dependent, with neuronal SGs displaying a particularly complex repertoire of proteins enriched in chaperones and autophagy factors. Strengthening the link between SGs and neurodegeneration, we demonstrate aberrant dynamics, composition, and subcellular distribution of SGs in cells from amyotrophic lateral sclerosis (ALS) patients. Using three Drosophila ALS/FTD models, we identify SG-associated modifiers of neurotoxicity in vivo. Altogether, our results highlight SG proteins as central to understanding and ultimately targeting neurodegeneration.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Grânulos Citoplasmáticos/metabolismo , Mapas de Interação de Proteínas , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Neurônios/metabolismo , Transporte ProteicoRESUMO
In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Grânulos Citoplasmáticos/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutagênese , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/genética , Prolina/análise , Prolina/metabolismo , Domínios Proteicos , Ribonucleases/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Estresse FisiológicoRESUMO
When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure. Here, we report that the ER luminal co-chaperone ERdj4/DNAJB9 is a selective IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). In vitro, ERdj4 is required for complex formation between BiP and IRE1LD. ERdj4 associates with IRE1LD and recruits BiP through the stimulation of ATP hydrolysis, forcibly disrupting IRE1 dimers. Unfolded proteins compete for BiP and restore IRE1LD to its default, dimeric, and active state. These observations establish BiP and its J domain co-chaperones as key regulators of the UPR.
Assuntos
Endorribonucleases/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Resposta a Proteínas não Dobradas , Animais , Cricetinae , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Humanos , Dobramento de ProteínaRESUMO
Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
Assuntos
Proteostase , Fatores de Transcrição , Proteostase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta ao Choque Térmico/genética , Chaperonas Moleculares/genética , Cromatina/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismoRESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Assuntos
Proteínas de Choque Térmico HSP70 , Neoplasias , Humanos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , RNA , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/genética , RNA não Traduzido/genéticaRESUMO
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genéticaRESUMO
Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.
Assuntos
Chaperonina 60/química , Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico Pequenas/química , Proteínas Mitocondriais/química , Desdobramento de Proteína , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Agregados Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Rhodospirillum rubrum/química , Rhodospirillum rubrum/metabolismoRESUMO
The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience in Caenorhabditis elegans We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Metabolismo dos Lipídeos , Proteostase , Receptores Citoplasmáticos e Nucleares , Reprodução , Transdução de Sinais , Estresse Fisiológico , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Metabolismo dos Lipídeos/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Reprodução/genética , Reprodução/fisiologia , Complexo Mediador/genética , Complexo Mediador/metabolismoRESUMO
More than 98% of the mammalian genome is noncoding, and interspersed transposable elements account for â¼50% of noncoding space. Here, we demonstrate that a specific interaction between the polycomb protein EZH2 and RNA made from B2 SINE retrotransposons controls stress-responsive genes in mouse cells. In the heat-shock model, B2 RNA binds stress genes and suppresses their transcription. Upon stress, EZH2 is recruited and triggers cleavage of B2 RNA. B2 degradation in turn upregulates stress genes. Evidence indicates that B2 RNA operates as a "speed bump" against advancement of RNA polymerase II, and temperature stress releases the brakes on transcriptional elongation. These data attribute a new function to EZH2 that is independent of its histone methyltransferase activity and reconcile how EZH2 can be associated with both gene repression and activation. Our study reveals that EZH2 and B2 together control activation of a large network of genes involved in thermal stress.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Resposta ao Choque Térmico , RNA não Traduzido/metabolismo , Retroelementos , Animais , Células-Tronco Embrionárias/metabolismo , Camundongos , Células NIH 3T3 , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
The conserved regulon of heat shock factor 1 in budding yeast contains chaperones for general protein folding as well as zinc-finger protein Zpr1, whose essential role in archaea and eukaryotes remains unknown. Here, we show that Zpr1 depletion causes acute proteotoxicity driven by biosynthesis of misfolded eukaryotic translation elongation factor 1A (eEF1A). Prolonged Zpr1 depletion leads to eEF1A insufficiency, thereby inducing the integrated stress response and inhibiting protein synthesis. Strikingly, we show by using two distinct biochemical reconstitution approaches that Zpr1 enables eEF1A to achieve a conformational state resistant to protease digestion. Lastly, we use a ColabFold model of the Zpr1-eEF1A complex to reveal a folding mechanism mediated by the Zpr1's zinc-finger and alpha-helical hairpin structures. Our work uncovers the long-sought-after function of Zpr1 as a bespoke chaperone tailored to the biogenesis of one of the most abundant proteins in the cell.
Assuntos
Proteínas de Transporte , Chaperonas Moleculares , Proteínas de Transporte/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biossíntese de Proteínas , Zinco/metabolismo , Dedos de Zinco , Fator 1 de Elongação de Peptídeos/metabolismoRESUMO
General protein folding is mediated by chaperones that utilize ATP hydrolysis to regulate client binding and release. Zinc-finger protein 1 (Zpr1) is an essential ATP-independent chaperone dedicated to the biogenesis of eukaryotic translation elongation factor 1A (eEF1A), a highly abundant GTP-binding protein. How Zpr1-mediated folding is regulated to ensure rapid Zpr1 recycling remains an unanswered question. Here, we use yeast genetics and microscopy analysis, biochemical reconstitution, and structural modeling to reveal that folding of eEF1A by Zpr1 requires GTP hydrolysis. Furthermore, we identify the highly conserved altered inheritance of mitochondria 29 (Aim29) protein as a Zpr1 co-chaperone that recognizes eEF1A in the GTP-bound, pre-hydrolysis conformation. This interaction dampens Zpr1â eEF1A GTPase activity and facilitates client exit from the folding cycle. Our work reveals that a bespoke ATP-independent chaperone system has mechanistic similarity to ATPase chaperones but unexpectedly relies on client GTP hydrolysis to regulate the chaperone-client interaction.
Assuntos
Proteínas de Transporte , GTP Fosfo-Hidrolases , Chaperonas Moleculares , Fatores de Alongamento de Peptídeos , Proteínas de Saccharomyces cerevisiae , Humanos , Trifosfato de Adenosina , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato , Chaperonas Moleculares/genética , Fatores de Alongamento de Peptídeos/metabolismo , Saccharomyces cerevisiae , Proteínas de Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Dobramento de ProteínaRESUMO
Eusocial insect reproductive females show strikingly longer life spans than nonreproductive female workers despite high genetic similarity. In the ant Harpegnathos saltator (Hsal), workers can transition to reproductive "gamergates," acquiring a fivefold prolonged life span by mechanisms that are poorly understood. We found that gamergates have elevated expression of heat shock response (HSR) genes in the absence of heat stress and enhanced survival with heat stress. This HSR gene elevation is driven in part by gamergate-specific up-regulation of the gene encoding a truncated form of a heat shock factor most similar to mammalian HSF2 (hsalHSF2). In workers, hsalHSF2 was bound to DNA only upon heat stress. In gamergates, hsalHSF2 bound to DNA even in the absence of heat stress and was localized to gamergate-biased HSR genes. Expression of hsalHSF2 in Drosophila melanogaster led to enhanced heat shock survival and extended life span in the absence of heat stress. Molecular characterization illuminated multiple parallels between long-lived flies and gamergates, underscoring the centrality of hsalHSF2 to extended ant life span. Hence, ant caste-specific heat stress resilience and extended longevity can be transferred to flies via hsalHSF2. These findings reinforce the critical role of proteostasis in health and aging and reveal novel mechanisms underlying facultative life span extension in ants.
Assuntos
Formigas , Longevidade , Animais , Feminino , Longevidade/genética , Formigas/genética , Drosophila melanogaster/genética , Envelhecimento , Resposta ao Choque Térmico/genética , MamíferosRESUMO
Stresses such as heat shock trigger the formation of protein aggregates and the induction of a disaggregation system composed of molecular chaperones. Recent work reveals that several cases of apparent heat-induced aggregation, long thought to be the result of toxic misfolding, instead reflect evolved, adaptive biomolecular condensation, with chaperone activity contributing to condensate regulation. Here we show that the yeast disaggregation system directly disperses heat-induced biomolecular condensates of endogenous poly(A)-binding protein (Pab1) orders of magnitude more rapidly than aggregates of the most commonly used misfolded model substrate, firefly luciferase. Beyond its efficiency, heat-induced condensate dispersal differs from heat-induced aggregate dispersal in its molecular requirements and mechanistic behavior. Our work establishes a bona fide endogenous heat-induced substrate for long-studied heat shock proteins, isolates a specific example of chaperone regulation of condensates, and underscores needed expansion of the proteotoxic interpretation of the heat shock response to encompass adaptive, chaperone-mediated regulation.
Assuntos
Condensados Biomoleculares/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ligação Competitiva , Condensados Biomoleculares/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Proteínas de Ligação a Poli(A)/genética , Agregados Proteicos , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, whereas an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become constitutively active without intergenic coalescence, which comes at a fitness cost. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.
Assuntos
Resposta ao Choque Térmico , Corpos Nucleares , Animais , Resposta ao Choque Térmico/genética , Proteínas de Choque Térmico HSP70/genética , Mamíferos , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , GenomaRESUMO
The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.
Assuntos
Poliadenilação , Transcriptoma , Resposta ao Choque Térmico/genética , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genéticaRESUMO
Fever, an evolutionarily conserved physiological response to infection, is also commonly associated with many autoimmune diseases, but its role in T cell differentiation and autoimmunity remains largely unclear. T helper 17 (Th17) cells are critical in host defense and autoinflammatory diseases, with distinct phenotypes and pathogenicity. Here, we show that febrile temperature selectively regulated Th17 cell differentiation in vitro in enhancing interleukin-17 (IL-17), IL-17F, and IL-22 expression. Th17 cells generated under febrile temperature (38.5°C-39.5°C), compared with those under 37°C, showed enhanced pathogenic gene expression with increased pro-inflammatory activities in vivo. Mechanistically, febrile temperature promoted SUMOylation of SMAD4 transcription factor to facilitate its nuclear localization; SMAD4 deficiency selectively abrogated the effects of febrile temperature on Th17 cell differentiation both in vitro and ameliorated an autoimmune disease model. Our results thus demonstrate a critical role of fever in shaping adaptive immune responses with implications in autoimmune diseases.