RESUMO
The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.
Assuntos
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , ProteostaseRESUMO
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Família Multigênica , Nostoc/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Galinhas , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Evolução Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Lipossomas Unilamelares/metabolismoRESUMO
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
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
The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano et al.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.
Assuntos
Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Biossíntese de Proteínas , RNA Mensageiro , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Humanos , Animais , Conformação Proteica , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genéticaRESUMO
Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes-induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.
Assuntos
Listeria monocytogenes/fisiologia , Proteínas Mitocondriais/fisiologia , Mitofagia , Animais , Autofagia , Toxinas Bacterianas/metabolismo , Linhagem Celular , Feminino , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Humanos , Listeria monocytogenes/patogenicidade , Listeriose/metabolismo , Listeriose/microbiologia , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Viabilidade Microbiana , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Fatores de Virulência/metabolismoRESUMO
Caloric restriction (CR) extends the lifespan of flies, worms, and yeast by counteracting age-related oxidation of H2O2-scavenging peroxiredoxins (Prxs). Here, we show that increased dosage of the major cytosolic Prx in yeast, Tsa1, extends lifespan in an Hsp70 chaperone-dependent and CR-independent manner without increasing H2O2 scavenging or genome stability. We found that Tsa1 and Hsp70 physically interact and that hyperoxidation of Tsa1 by H2O2 is required for the recruitment of the Hsp70 chaperones and the Hsp104 disaggregase to misfolded and aggregated proteins during aging, but not heat stress. Tsa1 counteracted the accumulation of ubiquitinated aggregates during aging and the reduction of hyperoxidized Tsa1 by sulfiredoxin facilitated clearance of H2O2-generated aggregates. The data reveal a conceptually new role for H2O2 signaling in proteostasis and lifespan control and shed new light on the selective benefits endowed to eukaryotic peroxiredoxins by their reversible hyperoxidation.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Peróxido de Hidrogênio/metabolismo , Longevidade , Peroxidases/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Restrição Calórica , Instabilidade Genômica , Proteínas de Choque Térmico/metabolismo , Humanos , Oxirredução , Agregados Proteicos , Saccharomyces cerevisiae/citologia , Transdução de SinaisRESUMO
Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1ß and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1ß-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Choque Térmico/metabolismo , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Choque Térmico/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A comprehensive analysis uncovered a set of yeast proteins promoting protein-based inheritance that shares many of the non-Mendelian properties of prions. Lacking any sequence or structural signatures of known prions, these proteins represent a new class of non-amyloid, protein-based epigenetic determinants that can control phenotype without impacting genotype.
Assuntos
Epigênese Genética , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Proteínas de Choque Térmico , Hereditariedade , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Príons/química , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; â¼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naive cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Thus, our data establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Característica Quantitativa Herdável , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Amiloide/metabolismo , Evolução Molecular , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Fases de Leitura Aberta , Príons/química , Príons/metabolismo , Proteoma , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms involving α-synuclein disaggregation or detoxification of soluble α-synuclein conformers. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinson's disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.
Assuntos
Doenças Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Animais , Humanos , alfa-Sinucleína/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismoRESUMO
Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation1,2. The mechanism that senses cellular temperature for inducing thermotolerance is still unclear3. Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrinsicamente Desordenadas , Temperatura , Sensação Térmica , Termotolerância , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sensação Térmica/genética , Sensação Térmica/fisiologia , Termotolerância/genética , Termotolerância/fisiologia , Fatores de Transcrição/metabolismo , Transdução de SinaisRESUMO
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
Mitogen-activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and coordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin-MAPK activation complex (sMAC)). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs allowed only partial functional recovery. T cells from old humans (>65 years old) or mice (16-20 months old) were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during aging.
Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/fisiologia , Proteínas de Choque Térmico/metabolismo , Imunidade , Imunossenescência , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Choque Térmico/genética , Humanos , Imunidade/genética , Imunossenescência/genética , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Interferente Pequeno/genética , Transdução de Sinais , Adulto JovemRESUMO
There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP-43, FUS, and α-synuclein proteotoxicity by mutating single residues in helix 1, 2, or 3 of the middle domain or the small domain of nucleotide-binding domain 1. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease-associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration.
Assuntos
Caenorhabditis elegans , Modelos Animais de Doenças , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/química , Humanos , Modelos Moleculares , Mutagênese , Neurônios/citologia , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Dobramento de Proteína , Estrutura Terciária de Proteína , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Deficiências na Proteostase/terapia , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , alfa-Sinucleína/metabolismoRESUMO
Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here, we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino-acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a nonredundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/genética , Embrião de Mamíferos/citologia , Feminino , Fibroblastos/metabolismo , Técnicas de Introdução de Genes , Proteínas de Choque Térmico/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Nucleares/genética , Peroxidases , Gravidez , Alinhamento de Sequência , Inanição/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/metabolismoRESUMO
Protein damage segregates asymmetrically in dividing yeast cells, rejuvenating daughters at the expense of mother cells. Zhou et al. now show that newly synthesized proteins are particularly prone to aggregation and describe a mechanism that tethers aggregated proteins to mitochondria. This association constrains aggregate mobility, effectively retaining and sorting toxic aggregates away from younger cells.
Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismoRESUMO
The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. Cells often delay replication in the face of stressful conditions, but the underlying mechanisms remain incompletely defined. Here, we demonstrate in Caulobacter crescentus that proteotoxic stress induces a cell-cycle arrest by triggering the degradation of DnaA, the conserved replication initiator. A depletion of available Hsp70 chaperone, DnaK, either through genetic manipulation or heat shock, induces synthesis of the Lon protease, which can directly degrade DnaA. Unexpectedly, we find that unfolded proteins, which accumulate following a loss of DnaK, also allosterically activate Lon to degrade DnaA, thereby ensuring a cell-cycle arrest. Our work reveals a mechanism for regulating DNA replication under adverse growth conditions. Additionally, our data indicate that unfolded proteins can actively and directly alter substrate recognition by cellular proteases.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/fisiologia , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Protease La/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Fator sigma/metabolismo , Estresse FisiológicoRESUMO
The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.