Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 56
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Mol Syst Biol ; 17(7): e10253, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34292675

RESUMEN

First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. Yet, the theory of model-based chassis design has rarely been put to rigorous experimental test. Here, we report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome-scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product-specific TCA cycle disruptions resulted in the secretion of the corresponding acid. Adaptive laboratory evolution further improved production of succinate and fumarate, demonstrating the evolutionary robustness of the engineered cells. In the case of malate, multi-omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. In all three cases, flux balance analysis integrating transcriptomics, proteomics and metabolomics data confirmed the flux re-routing predicted by the model. Taken together, our modelling and experimental results have implications for the computer-aided design of microbial cell factories.


Asunto(s)
Ingeniería Metabólica , Saccharomyces cerevisiae , Ciclo del Ácido Cítrico/genética , Metabolómica , Saccharomyces cerevisiae/genética , Ácido Succínico
2.
EMBO J ; 39(9): e103788, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-32064661

RESUMEN

Ribosome recycling by the twin-ATPase ABCE1 is a key regulatory process in mRNA translation and surveillance and in ribosome-associated protein quality control in Eukarya and Archaea. Here, we captured the archaeal 30S ribosome post-splitting complex at 2.8 Å resolution by cryo-electron microscopy. The structure reveals the dynamic behavior of structural motifs unique to ABCE1, which ultimately leads to ribosome splitting. More specifically, we provide molecular details on how conformational rearrangements of the iron-sulfur cluster domain and hinge regions of ABCE1 are linked to closure of its nucleotide-binding sites. The combination of mutational and functional analyses uncovers an intricate allosteric network between the ribosome, regulatory domains of ABCE1, and its two structurally and functionally asymmetric ATP-binding sites. Based on these data, we propose a refined model of how signals from the ribosome are integrated into the ATPase cycle of ABCE1 to orchestrate ribosome recycling.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Subunidades Ribosómicas Pequeñas de Archaea/metabolismo , Thermococcus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Microscopía por Crioelectrón , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Subunidades Ribosómicas Pequeñas de Archaea/química , Ribosomas/metabolismo , Thermococcus/genética
3.
Sci Rep ; 10(1): 895, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31964902

RESUMEN

The yeast fatty acid synthase (FAS) is a barrel-shaped 2.6 MDa complex. Upon barrel-formation, two multidomain subunits, each more than 200 kDa large, intertwine to form a heterododecameric complex that buries 170,000 Å2 of protein surface. In spite of the rich knowledge about yeast FAS in structure and function, its assembly remained elusive until recently, when co-translational interaction of the ß-subunit with the nascent α-subunit was found to initiate assembly. Here, we characterize the co-translational assembly of yeast FAS at a molecular level. We show that the co-translationally formed interface is sensitive to subtle perturbations, so that the exchange of two amino acids located in the emerging interface can prevent assembly. On the other hand, assembly can also be initiated via the co-translational interaction of the subunits at other sites, which implies that this process is not strictly site or sequence specific. We further highlight additional steps in the biogenesis of yeast FAS, as the formation of a dimeric subunit that orchestrates complex formation and acts as platform for post-translational phosphopantetheinylation. The presented data supports the understanding of the recently discovered prevalence of eukaryotic complexes for co-translational assembly, and is valuable for further harnessing FAS in the biotechnological production of aliphatic compounds.


Asunto(s)
Ácido Graso Sintasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Transportadora de Acilo/química , Ácido Graso Sintasas/química , Ácido Graso Sintasas/genética , Complejos Multienzimáticos/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Dominios Proteicos , Multimerización de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
4.
Nucleic Acids Res ; 48(3): 1435-1450, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31863583

RESUMEN

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.


Asunto(s)
Transferasas Alquil y Aril/genética , Proteínas Bacterianas/genética , ARN de Transferencia/genética , Transferasas Alquil y Aril/química , Proteínas Bacterianas/química , Escherichia coli/enzimología , Escherichia coli/genética , Conformación de Ácido Nucleico , Nucleótidos , Saccharomyces cerevisiae/enzimología
5.
Acta Neuropathol ; 138(6): 1053-1074, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31428936

RESUMEN

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Epigénesis Genética , Glioma/metabolismo , Metiltransferasas/metabolismo , Proteínas Musculares/metabolismo , Biosíntesis de Proteínas/fisiología , Ribosomas/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Metilación de ADN , Humanos , Metiltransferasas/genética , Ratones Desnudos , Proteínas Musculares/genética , Trasplante de Neoplasias , ARN Ribosómico 28S
6.
Appl Environ Microbiol ; 85(11)2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-30952662

RESUMEN

Lantibiotics subtilin and nisin are produced by Bacillus subtilis and Lactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)-mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducing B. subtilis strain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.IMPORTANCE The two lantibiotics nisin and subtilin are produced by Lactococcus lactis and Bacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.


Asunto(s)
Bacillus subtilis/inmunología , Bacillus subtilis/metabolismo , Bacteriocinas/metabolismo , Inmunidad , Nisina/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Bacteriocinas/genética , Farmacorresistencia Bacteriana , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Lactococcus lactis , Lipoproteínas/genética , Lipoproteínas/inmunología , Lipoproteínas/aislamiento & purificación , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo
7.
Sci Rep ; 8(1): 11904, 2018 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-30093689

RESUMEN

The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.


Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Adenosina/metabolismo , Secuencia de Bases , Electroforesis en Gel Bidimensional , Células HCT116 , Humanos , Metilación , Metiltransferasas/genética , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Dominios Proteicos , Proteína O-Metiltransferasa , Proteómica/métodos , ARN Ribosómico/química , ARN Ribosómico/genética , Proteínas de Unión al ARN , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Grandes/química , Subunidades Ribosómicas Grandes/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Appl Environ Microbiol ; 83(18)2017 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-28710266

RESUMEN

Autoinduction via two-component systems is a widespread regulatory mechanism that senses environmental and metabolic changes. Although the lantibiotics nisin and subtilin are closely related and share the same lanthionine ring structure, they autoinduce their biosynthesis in a highly specific manner. Subtilin activates only the two-component system SpaRK of Bacillus subtilis, whereas nisin activates solely the two-component system NisRK of Lactococcus lactis To identify components that determine the specificity of subtilin autoinduction, several variants of the respective lantibiotics were analyzed for their autoinductive capacities. Here, we show that amino acid position 20 is crucial for SpaK activation, as an engineered nisin molecule with phenylalanine at position 20 (nisin N20F) was able to activate SpaK in a specific manner. In combination with the N-terminal tryptophan of subtilin (nisin I1W/N20F), SpaK autoinduction reached almost the level of subtilin-mediated autoinduction. Furthermore, the overall structure of subtilin is also important for its association with the histidine kinase. The destruction of the second lanthionine ring (subtilin C11A, ring B), as well as mutations that interfere with the flexibility of the hinge region located between lanthionine rings C and D (subtilin L21P/Q22P), abolished SpaK autoinduction. Although the C-terminal part of subtilin is needed for efficient SpaK autoinduction, the destruction of lanthionine rings D and E had no measurable impact. Based on these findings, a model for the interaction of subtilin with histidine kinase SpaK was established.IMPORTANCE Although two-component systems are important regulatory systems that sense environmental changes, very little information on the molecular mechanism of sensing or the interaction of the sensor with its respective kinase is available. The strong specificity of linear lantibiotics such as subtilin and nisin for their respective kinases provides an excellent model system to unravel the structural needs of these lantibiotics for activating histidine kinases in a specific manner. More than that, the biosyntheses of lantibiotics are autoinduced via two-component systems. Therefore, an understanding of their interactions with histidine kinases is needed for the biosynthesis of newly engineered peptide antibiotics. Using a Bacillus subtilis-based reporter system, we were able to identify the molecular constraints that are necessary for specific SpaK activation and to provide SpaK specificity to nisin with just two point mutations.

9.
PLoS Genet ; 13(5): e1006804, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28542199

RESUMEN

Box C/D snoRNAs are known to guide site-specific ribose methylation of ribosomal RNA. Here, we demonstrate a novel and unexpected role for box C/D snoRNAs in guiding 18S rRNA acetylation in yeast. Our results demonstrate, for the first time, that the acetylation of two cytosine residues in 18S rRNA catalyzed by Kre33 is guided by two orphan box C/D snoRNAs-snR4 and snR45 -not known to be involved in methylation in yeast. We identified Kre33 binding sites on these snoRNAs as well as on the 18S rRNA, and demonstrate that both snR4 and snR45 establish extended bipartite complementarity around the cytosines targeted for acetylation, similar to pseudouridylation pocket formation by the H/ACA snoRNPs. We show that base pairing between these snoRNAs and 18S rRNA requires the putative helicase activity of Kre33, which is also needed to aid early pre-rRNA processing. Compared to yeast, the number of orphan box C/D snoRNAs in higher eukaryotes is much larger and we hypothesize that several of these may be involved in base-modifications.


Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/metabolismo , ARN Nuclear Pequeño/metabolismo , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Sitios de Unión , Citosina/metabolismo , Unión Proteica , ARN Ribosómico 18S/genética , ARN Nuclear Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Nat Struct Mol Biol ; 24(5): 453-460, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28368393

RESUMEN

The essential ATP-binding cassette protein ABCE1 splits 80S ribosomes into 60S and 40S subunits after canonical termination or quality-control-based mRNA surveillance processes. However, the underlying splitting mechanism remains enigmatic. Here, we present a cryo-EM structure of the yeast 40S-ABCE1 post-splitting complex at 3.9-Å resolution. Compared to the pre-splitting state, we observe repositioning of ABCE1's iron-sulfur cluster domain, which rotates 150° into a binding pocket on the 40S subunit. This repositioning explains a newly observed anti-association activity of ABCE1. Notably, the movement implies a collision with A-site factors, thus explaining the splitting mechanism. Disruption of key interactions in the post-splitting complex impairs cellular homeostasis. Additionally, the structure of a native post-splitting complex reveals ABCE1 to be part of the 43S initiation complex, suggesting a coordination of termination, recycling, and initiation.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/ultraestructura , Microscopía por Crioelectrón , Modelos Biológicos , Modelos Moleculares , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , Unión Proteica , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura
11.
PLoS One ; 12(3): e0173940, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28278232

RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0168873.].

12.
PLoS One ; 11(12): e0168873, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-28033325

RESUMEN

Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.


Asunto(s)
Proteínas de Plantas/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosa/metabolismo , Saccharomyces cerevisiae/genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Secuencia de Bases , Cromatografía de Fase Inversa , Metilación , Mutación Puntual , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Ribosomas/genética , Ribosomas/metabolismo
13.
Biochim Biophys Acta ; 1862(9): 1558-69, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27240544

RESUMEN

Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.


Asunto(s)
Ataxina-2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Arsenitos/toxicidad , Ataxina-2/antagonistas & inhibidores , Ataxina-2/genética , Línea Celular Tumoral , Células Cultivadas , Factores Eucarióticos de Iniciación/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Ratones , Neuronas/metabolismo , Fosforilación , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteína S6 Ribosómica/metabolismo , Inanición/genética , Inanición/metabolismo , Estrés Fisiológico
14.
Nucleic Acids Res ; 44(9): 4304-16, 2016 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-27084949

RESUMEN

The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m(1)acp(3)Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.


Asunto(s)
Transferasas Alquil y Aril/fisiología , ARN Ribosómico 18S/biosíntesis , Saccharomyces cerevisiae/enzimología , Transferasas Alquil y Aril/química , Dominio Catalítico , Cristalografía por Rayos X , Células HCT116 , Humanos , Enlace de Hidrógeno , Secuencias Invertidas Repetidas , Modelos Moleculares , Unión Proteica , Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/química , S-Adenosilmetionina/química
15.
FEMS Yeast Res ; 16(3)2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26895788

RESUMEN

Pyruvate and acetyl-coenzyme A, located at the interface between glycolysis and TCA cycle, are important intermediates in yeast metabolism and key precursors for industrially relevant products. Rational engineering of their supply requires knowledge of compensatory reactions that replace predominant pathways when these are inactivated. This study investigates effects of individual and combined mutations that inactivate the mitochondrial pyruvate-dehydrogenase (PDH) complex, extramitochondrial citrate synthase (Cit2) and mitochondrial CoA-transferase (Ach1) in Saccharomyces cerevisiae. Additionally, strains with a constitutively expressed carnitine shuttle were constructed and analyzed. A predominant role of the PDH complex in linking glycolysis and TCA cycle in glucose-grown batch cultures could be functionally replaced by the combined activity of the cytosolic PDH bypass and Cit2. Strongly impaired growth and a high incidence of respiratory deficiency in pda1Δ ach1Δ strains showed that synthesis of intramitochondrial acetyl-CoA as a metabolic precursor requires activity of either the PDH complex or Ach1. Constitutive overexpression of AGP2, HNM1, YAT2, YAT1, CRC1 and CAT2 enabled the carnitine shuttle to efficiently link glycolysis and TCA cycle in l-carnitine-supplemented, glucose-grown batch cultures. Strains in which all known reactions at the glycolysis-TCA cycle interface were inactivated still grew slowly on glucose, indicating additional flexibility at this key metabolic junction.


Asunto(s)
Ciclo del Ácido Cítrico , Glucólisis , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Citrato (si)-Sintasa/genética , Coenzima A Transferasas/genética , Eliminación de Gen , Expresión Génica , Ingeniería Metabólica , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas/genética , Complejo Piruvato Deshidrogenasa/genética , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética
16.
J Biol Chem ; 290(48): 28869-86, 2015 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-26459561

RESUMEN

Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.


Asunto(s)
Proteínas Bacterianas/química , Bacteriocinas/química , Lactococcus lactis/química , Lipoproteínas/química , Proteínas de la Membrana/química , Nisina/química , Bacillus subtilis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Nisina/genética , Nisina/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-Actividad
17.
Appl Environ Microbiol ; 81(22): 7914-23, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26341212

RESUMEN

The biosynthesis of the lantibiotics subtilin and nisin is regulated by autoinduction via two-component systems. Although subtilin is structurally closely related to nisin and contains the same lanthionine ring structure, both lantibiotics specifically autoinduce their biosynthesis. Subtilin and also the subtilin-like lantibiotics entianin and ericin autoinduce the two-component system SpaRK of Bacillus subtilis, whereas the biosynthesis of nisin is autoinduced via the two-component system NisRK of Lactococcus lactis. Autoinduction is highly specific for the respective lantibiotic and therefore of major importance for the functional expression of genetically engineered subtilin-like lantibiotics. To identify the structural features required for subtilin autoinduction, subtilin-nisin hybrids and specific point mutations of amino acid position 1 were generated. For subtilin autoinduction, the N-terminal tryptophan is the most important for full SpaK activation. The failure of subtilin to autoinduce the histidine kinase NisK mainly depends on the N-terminal tryptophan, as its single exchange to the aliphatic amino acid residues isoleucine, leucine, and valine provided NisK autoinduction. In addition, the production of subtilin variants which did not autoinduce their own biosynthesis could be rescued upon heterologous coexpression in B. subtilis DSM15029 by the autoinducing subtilin-like lantibiotic entianin.


Asunto(s)
Bacillus subtilis/genética , Bacteriocinas/genética , Regulación Bacteriana de la Expresión Génica , Nisina/genética , Bacillus subtilis/metabolismo , Bacteriocinas/metabolismo , Nisina/metabolismo , Análisis de Secuencia de ADN
18.
Nat Chem Biol ; 11(9): 625-31, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26284661
19.
Appl Environ Microbiol ; 81(16): 5335-43, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26025904

RESUMEN

The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1-20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1-20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1-20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism.


Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Bacteriocinas/metabolismo , Proteínas Quinasas/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Activación Enzimática , Histidina Quinasa , Nisina/metabolismo , Uridina Difosfato Ácido N-Acetilmurámico/metabolismo
20.
Cell Rep ; 10(7): 1215-25, 2015 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-25704822

RESUMEN

Mitophagy is crucial to ensuring mitochondrial quality control. However, the molecular mechanism and regulation of mitophagy are still not fully understood. Here, we developed a quantitative methodology termed synthetic quantitative array (SQA) technology, which allowed us to perform a genome-wide screen for modulators of rapamycin-induced mitophagy in S. cerevisiae. SQA technology can be easily employed for other enzyme-based reporter systems and widely applied in yeast research. We identified 86 positive and 10 negative regulators of mitophagy. Moreover, SQA-based analysis of non-selective autophagy revealed that 63 of these regulators are specific for mitophagy and 33 regulate autophagy in general. The Ubp3-Bre5 deubiquitination complex was found to inhibit mitophagy but, conversely, to promote other types of autophagy, including ribophagy. This complex translocates dynamically to mitochondria upon induction of mitophagy. These findings point to a role of ubiquitination in mitophagy in yeast and suggest a reciprocal regulation of distinct autophagy pathways.


Asunto(s)
Endopeptidasas/metabolismo , Mitocondrias/metabolismo , Mitofagia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia/efectos de los fármacos , Endopeptidasas/genética , Genoma Fúngico , Mitofagia/efectos de los fármacos , Mutagénesis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sirolimus/farmacología , Ubiquitinación
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA