RESUMEN
CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.
Asunto(s)
Bacterias , Bacteriófagos , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Nucleótidos Cíclicos , Proteasa La , Bacterias/enzimología , Bacterias/inmunología , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/inmunología , Bacteriófagos/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Activación Enzimática , Regulación Bacteriana de la Expresión Génica , Nucleótidos Cíclicos/inmunología , Nucleótidos Cíclicos/metabolismo , Operón , Proteasa La/química , Proteasa La/metabolismo , ARN Viral , Factor sigma , Transcripción GenéticaRESUMEN
Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Polifosfatos/metabolismo , Proteasa La/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/citología , Caulobacter crescentus/fisiología , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteasa La/metabolismo , Proteínas Bacterianas/genética , Caulobacter crescentus/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Factor sigma/metabolismo , Estrés FisiológicoRESUMEN
During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.
Asunto(s)
Caulobacter crescentus/metabolismo , Nucleótidos/metabolismo , Proteasa La/metabolismo , Pliegue de Proteína , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/enzimología , Elementos Transponibles de ADN , Didesoxinucleósidos/metabolismo , Regulación Bacteriana de la Expresión Génica , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Ribonucleótido Reductasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
All cells use proteases to adjust protein amounts. Proteases maintain protein homeostasis by degrading nonfunctional toxic proteins and play regulatory roles by targeting particular substrates in response to specific signals. Here we address how cells tune protease specificity to nutritional signals. We report that Salmonella enterica increases the specificity of the broadly conserved proteases Lon and ClpSAP by transforming the Lon activator and substrate HspQ into an inhibitor of the N-degron recognin ClpS, the adaptor of the ClpAP protease. We establish that upon acetylation, HspQ stops being a Lon activator and substrate and that the accumulated HspQ binds to ClpS, hindering degradation of ClpSAP substrates. Growth on glucose promotes HspQ acetylation by increasing acetyl-CoA amounts, thereby linking metabolism to proteolysis. By altering protease specificities but continuing to degrade junk proteins, cells modify the abundance of particular proteins while preserving the quality of their proteomes. This rapid response mechanism linking protease specificity to nutritional signals is broadly conserved.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fenómenos Fisiológicos de la Nutrición , Salmonella enterica/enzimología , Acetilación , Activadores de Enzimas/metabolismo , Inhibidores Enzimáticos/metabolismo , Glucosa/metabolismo , Proteínas de Choque Térmico , Proteasa La/metabolismo , Unión Proteica , Proteolisis , Salmonella enterica/crecimiento & desarrollo , Especificidad por SustratoRESUMEN
RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Mutación , Fenotipo , Ribonucleasa P , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Proteasa La/genética , Proteasa La/metabolismo , Supresión Genética , TemperaturaRESUMEN
The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism; however, the function and regulation of elevated mitochondrial metabolism in EC in response to proinflammatory cytokines remain unclear. Studies using high-resolution metabolomics and 13C-glucose and 13C-glutamine labeling flux techniques showed that pyruvate dehydrogenase activity (PDH) and oxidative tricarboxylic acid cycle (TCA) flux are elevated in human umbilical vein ECs in response to overnight (16 h) treatment with TNFα (10 ng/mL). Mechanistic studies indicated that TNFα mediated these metabolic changes via mitochondrial-specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-κB-dependent mechanism. Using RNA sequencing following siRNA-mediated knockdown of the catalytically active subunit of PDH, PDHE1α (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are up-regulated by TNFα stimulation. Notably, TNFα-induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Metabolomics and ChIP sequencing for acetylated modification on lysine 27 of histone 3 (H3K27ac) showed that TNFα-induced PDH flux promotes histone acetylation of specific gene loci via citrate accumulation and ATP-citrate lyase-mediated generation of acetyl CoA. Together, these results uncover a mechanism by which TNFα signaling increases oxidative TCA flux of glucose to support TNFα-induced gene transcription through extramitochondrial acetyl CoA generation and histone acetylation.
Asunto(s)
Proteasa La , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Acetilcoenzima A , Células Endoteliales , Histonas , CitocinasRESUMEN
The mitochondrial matrix AAA+ Lon protease (LONP1) degrades misfolded or unassembled proteins, which play a pivotal role in mitochondrial quality control. During heart development, a metabolic shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation takes place, which relies strongly on functional mitochondria. However, the relationship between the mitochondrial quality control machinery and metabolic shifts is elusive. Here, we interfered with mitochondrial quality control by inactivating Lonp1 in murine embryonic cardiac tissue, resulting in severely impaired heart development, leading to embryonic lethality. Mitochondrial swelling, cristae loss and abnormal protein aggregates were evident in the mitochondria of Lonp1-deficient cardiomyocytes. Accordingly, the p-eIF2α-ATF4 pathway was triggered, and nuclear translocation of ATF4 was observed. We further demonstrated that ATF4 regulates the expression of Tfam negatively while promoting that of Glut1, which was responsible for the disruption of the metabolic shift to oxidative phosphorylation. In addition, elevated levels of reactive oxygen species were observed in Lonp1-deficient cardiomyocytes. This study revealed that LONP1 safeguards metabolic shifts in the developing heart by controlling mitochondrial protein quality, suggesting that disrupted mitochondrial quality control may cause prenatal cardiomyopathy.
Asunto(s)
Corazón , Mitocondrias Cardíacas , Proteasa La , Proteasas ATP-Dependientes/metabolismo , Animales , Corazón/crecimiento & desarrollo , Ratones , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Proteasa La/genética , Proteasa La/metabolismoRESUMEN
The AAA+ Lon protease is conserved from bacteria to humans, performs crucial roles in protein homeostasis, and is implicated in bacterial pathogenesis and human disease. We investigated how Lon selectively degrades specific substrates among a diverse array of potential targets. We report the discovery of HspQ as a new Lon substrate, unique specificity-enhancing factor, and potent allosteric activator. Lon recognizes HspQ via a C-terminal degron, whose precise presentation, in synergy with multipartite contacts with the native core of HspQ, is required for allosteric Lon activation. Productive HspQ-Lon engagement enhances degradation of multiple new and known Lon substrates. Our studies reveal the existence and simultaneous utilization of two distinct substrate recognition sites on Lon, an HspQ binding site and an HspQ-modulated allosteric site. Our investigations unveil an unprecedented regulatory use of an evolutionarily conserved heat shock protein and present a distinctive mechanism for how Lon protease achieves temporally enhanced substrate selectivity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteasa La/metabolismo , Yersinia pestis/enzimología , Regulación Alostérica , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas de Choque Térmico/genética , Cinética , Proteasa La/genética , Unión Proteica , Pliegue de Proteína , Proteolisis , Especificidad por Sustrato , Yersinia pestis/genéticaRESUMEN
Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.
Asunto(s)
Acinetobacter , Proteasa La , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Archaea/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , ADN/metabolismo , ADN Helicasas/metabolismo , Unión Proteica , Acinetobacter/enzimología , Acinetobacter/virología , Proteasa La/ultraestructuraRESUMEN
Horizontal gene transfer drives bacterial evolution. To confer new properties, horizontally acquired genes must overcome gene silencing by nucleoid-associated proteins, such as the heat-stable nucleoid structuring (H-NS) protein. Enteric bacteria possess proteins that displace H-NS from foreign genes, form nonfunctional oligomers with H-NS, and degrade H-NS, raising the question of whether any of these mechanisms play a role in overcoming foreign gene silencing in vivo. To answer this question, we mutagenized the hns gene and identified a variant specifying an H-NS protein that binds foreign DNA and silences expression of the corresponding genes, like wild-type H-NS, but resists degradation by the Lon protease. Critically, Escherichia coli expressing this variant alone fails to produce curli, which are encoded by foreign genes and required for biofilm formation, and fails to colonize the murine gut. Our findings establish that H-NS proteolysis is a general mechanism of derepressing foreign genes and essential for colonization of mammalian hosts.
Asunto(s)
Proteínas de Escherichia coli , Proteasa La , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Mamíferos/metabolismo , Ratones , Proteasa La/genética , Proteasa La/metabolismoRESUMEN
Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. IMPORTANCE: The Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.
Asunto(s)
Proteínas Bacterianas , Caulobacter crescentus , Regulación Bacteriana de la Expresión Génica , Proteasa La , Proteolisis , Transducción de Señal , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Caulobacter crescentus/enzimología , Caulobacter crescentus/crecimiento & desarrollo , Proteasa La/metabolismo , Proteasa La/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hemo/metabolismo , Histidina QuinasaRESUMEN
Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.
Asunto(s)
Proteínas de Escherichia coli , Proteasa La , Escherichia coli/genética , Fosforilación Oxidativa , Proteasa La/genética , Carbonil Cianuro m-Clorofenil Hidrazona , Aguas Residuales , Antibacterianos/farmacología , Dinitrofenoles , Proteínas de Escherichia coli/genéticaRESUMEN
IMPORTANCE: Regulated protein degradation is a critical process in all cell types, which contributes to the precise regulation of protein amounts in response to internal and external cues. In bacteria, protein degradation is carried out by ATP-dependent proteases. Although past work revealed detailed insights into the operation principles of these proteases, there is limited knowledge about the substrate proteins that are degraded by distinct proteases and the regulatory role of proteolysis in cellular processes. This study reveals a direct role of the conserved protease Lon in regulating σT, a transcriptional regulator of the general stress response in α-proteobacteria. Our work is significant as it underscores the importance of regulated proteolysis in modulating the levels of key regulatory proteins under changing conditions.
Asunto(s)
Caulobacter crescentus , Proteasa La , Proteolisis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteasa La/genética , Proteasa La/metabolismo , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades these unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphological perturbations. Although structures of the bacterial and human Lon protease reveal a hexameric assembly, yeast PIM1 was speculated to form a heptameric assembly and is uniquely characterized by a â¼50-residue insertion between the ATPase and protease domains. To further understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-electron microscopy structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrates to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a â¼15-residue C-terminal extension. These additional C-terminal residues form an α-helix located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1's enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.
Asunto(s)
Proteínas Mitocondriales , Proteasa La , Proteínas Proto-Oncogénicas c-pim-1 , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidasas , Proteasas ATP-Dependientes/metabolismo , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Péptido Hidrolasas/metabolismo , Proteasa La/química , Proteasa La/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/química , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Relación Estructura-ActividadRESUMEN
The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mutagénesis , Proteasa La/metabolismo , Control de Calidad , ARN Bacteriano/genética , Proteínas de Unión al ARN/metabolismo , Salmonella enterica/metabolismo , Proteínas Bacterianas/genética , Proteínas de Unión al ARN/genética , Salmonella enterica/genética , Salmonella enterica/crecimiento & desarrolloRESUMEN
BACKGROUND: Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae. OBJECTIVES: To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress. METHODS: The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics. RESULTS: A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex. CONCLUSIONS: Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.
Asunto(s)
Antitoxinas , Proteasa La , Antitoxinas/genética , Meropenem , Acetiltransferasas , Proteasa La/metabolismo , Operón , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
BACKGROUND: Sirtuin 3 (Sirt3) is a controversial regulator of carcinogenesis. It residents in the mitochondria and gradually decays during aging. In this study, we tried to investigate the role of Sirt3 in carcinogenesis and to explore its involvement in metabolic alteration. METHODS: We generated conditional intestinal epithelium Sirt3-knockout mice by crossing ApcMin/+; Villin-Cre with Sirt3fl/fl (AVS) mice. The deacetylation site of Lon protease-1 (LONP1) was identified with Mass spectrometry. The metabolic flux phenotype was determined by Seahorse bioanalyzer. RESULTS: We found that intestinal epithelial cell-specific ablation of Sirt3 promotes primary tumor growth via stabilizing mitochondrial LONP1. Notably, we newly identified that Sirt3 deacetylates human oncogene LONP1 at N terminal residue lysine 145 (K145). The LONP1 hyperacetylation-mutant K145Q enhances oxidative phosphorylation to accelerate tumor growth, whereas the deacetylation-mutant K145R produces calorie-restriction like phenotype to restrain tumorigenesis. Sirt3 deacetylates LONP1 at K145 and subsequently facilitates the ESCRT0 complex sorting and K63-ubiquitination that resulted in the degradation of LONP1. Our results sustain the notion that Sirt3 is a tumor-suppressor to maintain the appropriate ubiquitination and degradation of oncogene LONP1. CONCLUSION: Sirt3 represents a targetable metabolic checkpoint of oncogenesis, which produces energy restriction effects via maintaining LONP1 K145 deacetylation and subsequent K63 ubiquitination.
Asunto(s)
Neoplasias , Proteasa La , Sirtuina 3 , Animales , Humanos , Ratones , Acetilación , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , Transformación Celular Neoplásica , Proteínas Mitocondriales/genética , Proteasa La/genética , Proteasa La/metabolismo , Sirtuina 3/metabolismo , UbiquitinaciónRESUMEN
Hydrogen peroxide (H2O2) released from mitochondria regulates various cell signaling pathways. Given that H2O2-eliminating enzymes such as peroxiredoxin III (PrxIII) are abundant in mitochondria, however, it has remained unknown how such release can occur. Active PrxIII-SH undergoes reversible inactivation via hyperoxidation to PrxIII-SO2, which is then reduced by sulfiredoxin. We now show that the amounts of PrxIII-SO2 and sulfiredoxin undergo antiphasic circadian oscillation in the mitochondria of specific tissues of mice maintained under normal conditions. Cytosolic sulfiredoxin was found to be imported into the mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is promoted by H2O2 released from mitochondria. The imported sulfiredoxin is degraded by Lon in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of sulfiredoxin provide the basis for sulfiredoxin oscillation and consequent PrxIII-SO2 oscillation in mitochondria and likely result in an oscillatory H2O2 release.
Asunto(s)
Ritmo Circadiano , Mitocondrias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Animales , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Oxidación-Reducción , Peroxiredoxina III/metabolismo , Proteasa La/metabolismo , Transporte de Proteínas , Proteolisis , Dióxido de Azufre/metabolismo , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Mistranslation is typically deleterious for cells, although specific mistranslated proteins can confer a short-term benefit in a particular environment. However, given its large overall cost, the prevalence of high global mistranslation rates remains puzzling. Altering basal mistranslation levels of Escherichia coli in several ways, we show that generalized mistranslation enhances early survival under DNA damage, by rapidly activating the SOS response. Mistranslating cells maintain larger populations after exposure to DNA damage, and thus have a higher probability of sampling critical beneficial mutations. Both basal and artificially increased mistranslation increase the number of cells that are phenotypically tolerant and genetically resistant under DNA damage; they also enhance survival at high temperature. In contrast, decreasing the normal basal mistranslation rate reduces cell survival. This wide-ranging stress resistance relies on Lon protease, which is revealed as a key effector that induces the SOS response in addition to alleviating proteotoxic stress. The new links between error-prone protein synthesis, DNA damage, and generalised stress resistance indicate surprising coordination between intracellular stress responses and suggest a novel hypothesis to explain high global mistranslation rates.