RESUMO
The E3 ubiquitin-protein ligase TRIM21, of the RING-containing tripartite motif (TRIM) protein family, is a major autoantigen in autoimmune diseases and a modulator of innate immune signaling. Together with ubiquitin-conjugating enzyme E2 E1 (UBE2E1), TRIM21 acts both as an E3 ligase and as a substrate in autoubiquitination. We here report a 2.82-Å crystal structure of the human TRIM21 RING domain in complex with the human E2-conjugating UBE2E1 enzyme, in which a ubiquitin-targeted TRIM21 substrate lysine was captured in the UBE2E1 active site. The structure revealed that the direction of lysine entry is similar to that described for human proliferating cell nuclear antigen (PCNA), a small ubiquitin-like modifier (SUMO)-targeted substrate, and thus differs from the canonical SUMO-targeted substrate entry. In agreement, we found that critical UBE2E1 residues involved in the capture of the TRIM21 substrate lysine are conserved in ubiquitin-conjugating E2s, whereas residues critical for SUMOylation are not conserved. We noted that coordination of the acceptor lysine leads to remodeling of amino acid side-chain interactions between the UBE2E1 active site and the E2-E3 direct interface, including the so-called "linchpin" residue conserved in RING E3s and required for ubiquitination. The findings of our work support the notion that substrate lysine activation of an E2-E3-connecting allosteric path may trigger catalytic activity and contribute to the understanding of specific lysine targeting by ubiquitin-conjugating E2s.
Assuntos
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Estrutura Molecular , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ribonucleoproteínas/química , Alinhamento de Sequência , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/químicaRESUMO
Adaptation of S. aureus to the hostile environment of CF airways resulted in changed abundance of proteins involved in energy metabolism, cellular processes, transport and binding, but most importantly in an iron-scavenging phenotype and increased activity of superoxide dismutase M.
Assuntos
Fibrose Cística/microbiologia , Ferro/metabolismo , Sistema Respiratório/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Adaptação Fisiológica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Superóxido Dismutase/classificação , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.
Assuntos
Enzimas de Restrição-Modificação do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Biologia Sintética/métodosRESUMO
Fatty acid desaturases regulate the unsaturation status of cellular lipids. They comprise two distinct evolutionary lineages, a soluble class found in the plastids of higher plants and an integral membrane class found in plants, yeast (Saccharomyces cerevisiae), animals, and bacteria. Both classes exhibit a dimeric quaternary structure. Here, we test the functional significance of dimeric organization of the soluble castor Δ9-18:0-acyl carrier protein desaturase, specifically, the hypothesis that the enzyme uses an alternating subunit half-of-the-sites reactivity mechanism whereby substrate binding to one subunit is coordinated with product release from the other subunit. Using a fluorescence resonance energy transfer assay, we demonstrated that dimers stably associate at concentrations typical of desaturase assays. An active site mutant T104K/S202E, designed to occlude the substrate binding cavity, was expressed, purified, and its properties validated by x-ray crystallography, size exclusion chromatography, and activity assay. Heterodimers comprising distinctly tagged wild-type and inactive mutant subunits were purified at 1:1 stoichiometry. Despite having only one-half the number of active sites, purified heterodimers exhibit equivalent activity to wild-type homodimers, consistent with half-of-the-sites reactivity. However, because multiple rounds of turnover were observed, we conclude that substrate binding to one subunit is not required to facilitate product release from the second subunit. The observed half-of-the-sites reactivity could potentially buffer desaturase activity from oxidative inactivation. That soluble desaturases require only one active subunit per dimer for full activity represents a mechanistic difference from the membrane class of desaturases such as the Δ9-acyl-CoA, Ole1p, from yeast, which requires two catalytically competent subunits for activity.
Assuntos
Proteína de Transporte de Acila/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ricinus communis/enzimologia , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Proteínas Mutantes/isolamento & purificação , Mutação/genética , Multimerização Proteica , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host's immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC-MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes , Citosol/metabolismo , Staphylococcus aureus/metabolismo , Cromatografia Líquida , Espectrometria de MassasRESUMO
Surface proteins are important for the fitness and virulence of the Gram-positive pathogen Streptococcus pneumoniae. They are crucial for interaction of the pathogen with its human host during infection. Therefore, the analysis of the pneumococcal surface proteome is an important task that requires powerful tools. In this study, two different methods, an optimized biotinylation approach and shaving with trypsin beads, were applied to study the pneumococcal surface proteome and to identify surface-exposed protein domains, respectively. The identification of nearly 95% of the predicted lipoproteins and 75% of the predicted sortase substrates reflects the high coverage of the two classical surface protein classes accomplished in this study. Furthermore, the biotinylation approach was applied to study the impact of an impaired lipoprotein maturation pathway on the cell envelope proteome and exoproteome. Loss of the lipoprotein diacylglyceryl transferase Lgt leads to striking changes in the lipoprotein distribution. Many lipoproteins disappear from the surface proteome and accumulate in the exoproteome. Further insights into lipoprotein processing in pneumococci are provided by immunoblot analyses of bacterial lysates and corresponding supernatant fractions. Taken together, the first comprehensive overview of the pneumococcal surface and exoproteome is presented, and a model for lipoprotein processing in S. pneumoniae is proposed.
Assuntos
Proteínas de Bactérias/biossíntese , Lipoproteínas/biossíntese , Proteoma , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biotina/metabolismo , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Lipoproteínas/metabolismo , Reação em Cadeia da Polimerase , Frações Subcelulares/metabolismo , Tripsina/metabolismoRESUMO
The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.
Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Ciclo do Ácido Cítrico , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Regulação Bacteriana da Expressão Gênica , Gluconeogênese , Mutação , Estresse Oxidativo , Biossíntese de Proteínas , Proteólise , Proteínas Repressoras/antagonistas & inibidores , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Regiospecific desaturation of long-chain saturated fatty acids has been described as approaching the limits of the discriminatory power of enzymes because the substrate entirely lacks distinguishing features close to the site of dehydrogenation. To identify the elusive mechanism underlying regioselectivity, we have determined two crystal structures of the archetypal Δ9 desaturase from castor in complex with acyl carrier protein (ACP), which show the bound ACP ideally situated to position C9 and C10 of the acyl chain adjacent to the diiron active site for Δ9 desaturation. Analysis of the structures and modeling of the complex between the highly homologous ivy Δ4 desaturase and ACP, identified a residue located at the entrance to the binding cavity, Asp280 in the castor desaturase (Lys275 in the ivy desaturase), which is strictly conserved within Δ9 and Δ4 enzymes but differs between them. We hypothesized that interaction between Lys275 and the phosphate of the pantetheine, seen in the ivy model, is key to positioning C4 and C5 adjacent to the diiron center for Δ4 desaturation. Mutating castor Asp280 to Lys resulted in a major shift from Δ9 to Δ4 desaturation. Thus, interaction between desaturase side-chain 280 and phospho-serine 38 of ACP, approximately 27 Å from the site of double-bond formation, predisposes ACP binding that favors either Δ9 or Δ4 desaturation via repulsion (acidic side chain) or attraction (positively charged side chain), respectively. Understanding the mechanism underlying remote control of regioselectivity provides the foundation for reengineering desaturase enzymes to create designer chemical feedstocks that would provide alternatives to those currently obtained from petrochemicals.
Assuntos
Proteína de Transporte de Acila/metabolismo , Ácidos Graxos/metabolismo , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Conformação Proteica , Cristalização , Ácidos Graxos Dessaturases/metabolismo , Mutagênese , Estearoil-CoA Dessaturase , Especificidade por SubstratoRESUMO
A principally new type of an electrophoresis setup for the second dimension of 2DE named HPE (high performance electrophoresis) has recently become available that provides excellent reproducibility much superior to traditional 2DE. It takes up ideas from early beginnings of 2DE which could not be satisfactory realized at that time. The new HPE system is in contrast to all other established systems a horizontal electrophoresis that employs a new type of precast polyacrylamide gels on film-backing and runs on a multilevel flatbed electrophoresis apparatus. In a systematic approach we compared its features to traditional 2DE for the cytosolic proteome of Bacillus subtilis. Not only the reproducibility is enhanced, but also nearly all qualitative parameters as resolution, sensitivity, the number of protein spots (25% more), and the number of different proteins (also additional 25%) are markedly increased. More than 200 proteins were exclusively found in HPE. This new electrophoresis system does not use buffer tanks. No glass plates are needed. Therefore handling of gels is greatly facilitated and very simple to use even for personnel with low technical skills. The new HPE system is technically at the beginnings and further development with increased performance can be expected.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/análise , Eletroforese em Gel Bidimensional/métodos , Proteoma/análise , Resinas Acrílicas/química , Eletroforese em Gel de Poliacrilamida/métodos , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Owing to the low number of proteins necessary to render a bacterial cell viable, bacteria are extremely attractive model systems to understand how the genome sequence is translated into actual life processes. One of the most intensively investigated model organisms is Bacillus subtilis. It has attracted world-wide research interest, addressing cell differentiation and adaptation on a molecular scale as well as biotechnological production processes. Meanwhile, we are looking back on more than 25 years of B. subtilis proteomics. A wide range of methods have been developed during this period for the large-scale qualitative and quantitative proteome analysis. Currently, it is possible to identify and quantify more than 50% of the predicted proteome in different cellular subfractions. In this review, we summarize the development of B. subtilis proteomics during the past 25 years.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genoma Bacteriano , ProteômicaRESUMO
Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.
Assuntos
Proteínas de Bactérias/química , Ferro/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteoma/análise , Proteínas de Bactérias/metabolismo , Cromatografia Líquida/métodos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismoRESUMO
Plant plasma membrane (pm) vesicles from mycorrhizal tobacco (Nicotiana tabacum cv. Samsun) roots were isolated with negligible fungal contamination by the aqueous two-phase partitioning technique as proven by fatty acid analysis. Palmitvaccenic acid became apparent as an appropriate indicator for fungal membranes in root pm preparations. The pm vesicles had a low specific activity of the vanadate-sensitive ATPase and probably originated from non-infected root cells. In a phosphate-limited tobacco culture system, root colonisation by the vesicular arbuscular mycorrhizal fungus, Glomus mosseae, is inhibited by external nitrate in a dose-dependent way. However, detrimental high concentrations of 25 mM nitrate lead to the highest colonisation rate observed, indicating that the defence system of the plant is impaired. Nitric oxide formation by the pm-bound nitrite:NO reductase increased in parallel with external nitrate supply in mycorrhizal roots in comparison to the control plants, but decreased under excess nitrate. Mycorrhizal pm vesicles had roughly a twofold higher specific activity as the non-infected control plants when supplied with 10-15 mM nitrate.
Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Glomeromycota/fisiologia , Micorrizas/fisiologia , Nicotiana/efeitos dos fármacos , Nicotiana/microbiologia , Nitratos/farmacologia , Biomassa , Membrana Celular/enzimologia , Ácidos Graxos/metabolismo , Glomeromycota/efeitos dos fármacos , Micorrizas/efeitos dos fármacos , Nitratos/metabolismo , Nitrito Redutases/metabolismo , Oxirredutases/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Nicotiana/crescimento & desenvolvimentoRESUMO
The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.
Assuntos
Proteínas Inibidoras de Apoptose/química , Proteína Inibidora de Apoptose Neuronal/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Proteína 3 com Repetições IAP de Baculovírus , Cristalografia por Raios X , Humanos , Proteínas Inibidoras de Apoptose/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteína Inibidora de Apoptose Neuronal/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína LigasesRESUMO
We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Nucleotídeo Desaminases/química , Nucleotídeo Desaminases/metabolismo , Estrutura Terciária de Proteína , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Modelos Moleculares , Estrutura Molecular , NADP/química , NADP/metabolismo , Nucleotídeo Desaminases/genética , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Desidrogenase do Álcool de Açúcar/genéticaRESUMO
Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.
Assuntos
Eletroforese em Gel Bidimensional , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Bidimensional/métodos , Focalização Isoelétrica , Proteólise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tripsina , Fluxo de TrabalhoRESUMO
FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Cristalografia por Raios X , Escherichia coli , Fluorometria , Humanos , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Domínios Proteicos , TemperaturaRESUMO
The hydrothermal vent mussel Bathymodiolus azoricus lives in an intimate symbiosis with two types of chemosynthetic Gammaproteobacteria in its gills: a sulfur oxidizer and a methane oxidizer. Despite numerous investigations over the last decades, the degree of interdependence between the three symbiotic partners, their individual metabolic contributions, as well as the mechanism of carbon transfer from the symbionts to the host are poorly understood. We used a combination of proteomics and genomics to investigate the physiology and metabolism of the individual symbiotic partners. Our study revealed that key metabolic functions are most likely accomplished jointly by B. azoricus and its symbionts: (1) CO2 is pre-concentrated by the host for carbon fixation by the sulfur-oxidizing symbiont, and (2) the host replenishes essential biosynthetic TCA cycle intermediates for the sulfur-oxidizing symbiont. In return (3), the sulfur oxidizer may compensate for the host's putative deficiency in amino acid and cofactor biosynthesis. We also identified numerous 'symbiosis-specific' host proteins by comparing symbiont-containing and symbiont-free host tissues and symbiont fractions. These proteins included a large complement of host digestive enzymes in the gill that are likely involved in symbiont digestion and carbon transfer from the symbionts to the host.
Assuntos
Gammaproteobacteria/fisiologia , Mytilidae/microbiologia , Proteoma , Simbiose , Animais , Vias Biossintéticas , Brânquias/microbiologia , Fontes Hidrotermais , Metano/metabolismo , Mytilidae/genética , Oxirredução , Especificidade da Espécie , Enxofre/metabolismoRESUMO
Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tanquirases/antagonistas & inibidores , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células HEK293 , Humanos , Indazóis/química , Indazóis/farmacologia , Modelos Moleculares , Fenantrenos/química , Fenantrenos/farmacologia , Ftalazinas/química , Ftalazinas/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Tanquirases/metabolismoRESUMO
Hydroponically grown tobacco (Nicotiana tabacum L. cv. Samsun) roots exude proteases under non-stressed conditions. Ten different proteases could be distinguished by 2D-zymography of root exudate. The majority of the gelatinolytic activity was susceptible to serine protease inhibitors. One of the proteases could be assigned to an EST (SGN-P361478) by mass spectrometry of immune-purified root exudate. The sequence was completed by RACE-PCR and shows typical serine protease features of subtilase family S8A. Thermostability and SDS-insensitivity indicate a kinetically stable enzyme. Phylogenetic classification of this highly gelatinolytic subtilase showed SDD1 to be the closest relative in Arabidopsis thaliana (L. Heynh.). Even closer related protein sequences could be found in other distant plant genera indicating a high conservation of the subtilase. A 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase-like protein and suberisation-associated anionic peroxidase-like protein were co-immune-purified and identified by mass spectrometry and may constitute potential interaction partners.
RESUMO
MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members.