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1.
Nucleic Acids Res ; 52(4): 2012-2029, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38224450

RESUMO

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.


Assuntos
Imunidade Inata , Células Procarióticas , Sistema Imunitário , NAD+ Nucleosidase , Células Procarióticas/imunologia , RNA Guia de Sistemas CRISPR-Cas , Transdução de Sinais , Eucariotos/imunologia
2.
Biomacromolecules ; 23(3): 1195-1204, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35042326

RESUMO

Encapsulation of proteins can have advantages for their protection, stability, and delivery purposes. One of the options to encapsulate proteins is to incorporate them in complex coacervate core micelles (C3Ms). This can easily be achieved by mixing aqueous solutions of the protein and an oppositely charged neutral-hydrophilic diblock copolymer. However, protein-containing C3Ms often suffer from salt-inducible disintegration due to the low charge density of proteins. The aim of this study is to improve the salt stability of protein-containing C3Ms by increasing the net charge of the protein by tagging it with a charged polypeptide. As a model protein, we used CotA laccase and generated variants with 10, 20, 30, and 40 glutamic acids attached at the C-terminus of CotA using genetic engineering. Micelles were obtained by mixing the five CotA variants with poly(N-methyl-2-vinyl-pyridinium)-block-poly(ethylene oxide) (PM2VP128-b-PEO477) at pH 10.8. Hydrodynamic radii of the micelles of approximately 31, 27, and 23 nm for native CotA, CotA-E20, and CotA-E40, respectively, were determined using dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). The encapsulation efficiency was not affected using enzymes with a polyglutamic acid tail but resulted in more micelles with a smaller number of enzyme molecules per micelle. Furthermore, it was shown that the addition of a polyglutamic acid tail to CotA indeed resulted in improved salt stability of enzyme-containing C3Ms. Interestingly, the polyglutamic acid CotA variants showed an enhanced enzyme activity. This study demonstrates that increasing the net charge of enzymes through genetic engineering is a promising strategy to improve the practical applicability of C3Ms as enzyme delivery systems.


Assuntos
Micelas , Ácido Poliglutâmico , Peptídeos , Polietilenoglicóis/química , Polímeros/química , Cloreto de Sódio
3.
Arch Biochem Biophys ; 702: 108820, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33684360

RESUMO

4-Hydroxybenzoate 3-hydroxylase (PHBH) is the most extensively studied group A flavoprotein monooxygenase (FPMO). PHBH is almost exclusively found in prokaryotes, where its induction, usually as a consequence of lignin degradation, results in the regioselective formation of protocatechuate, one of the central intermediates in the global carbon cycle. In this contribution we introduce several less known FAD-dependent 4-hydroxybenzoate hydroxylases. Phylogenetic analysis showed that the enzymes discussed here reside in distinct clades of the group A FPMO family, indicating their separate divergence from a common ancestor. Protein homology modelling revealed that the fungal 4-hydroxybenzoate 3-hydroxylase PhhA is structurally related to phenol hydroxylase (PHHY) and 3-hydroxybenzoate 4-hydroxylase (3HB4H). 4-Hydroxybenzoate 1-hydroxylase (4HB1H) from yeast catalyzes an oxidative decarboxylation reaction and is structurally similar to 3-hydroxybenzoate 6-hydroxylase (3HB6H), salicylate hydroxylase (SALH) and 6-hydroxynicotinate 3-monooxygenase (6HNMO). Genome mining suggests that the 4HB1H activity is widespread in the fungal kingdom and might be responsible for the oxidative decarboxylation of vanillate, an import intermediate in lignin degradation. 4-Hydroxybenzoyl-CoA 1-hydroxylase (PhgA) catalyzes an intramolecular migration reaction (NIH shift) during the three-step conversion of 4-hydroxybenzoate to gentisate in certain Bacillus species. PhgA is phylogenetically related to 4-hydroxyphenylacetate 1-hydroxylase (4HPA1H). In summary, this paper shines light on the natural diversity of group A FPMOs that are involved in the aerobic microbial catabolism of 4-hydroxybenzoate.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Parabenos/metabolismo , Sequência de Aminoácidos , Hidroquinonas/metabolismo , Modelos Moleculares , Filogenia , Conformação Proteica
4.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298920

RESUMO

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.


Assuntos
Multimerização Proteica/fisiologia , Proteínas/metabolismo , Células Cultivadas , Citosol/metabolismo , Dictyostelium/metabolismo , Difusão , Dimerização , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Fótons , Espectrometria de Fluorescência/métodos
5.
Langmuir ; 36(29): 8494-8502, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32598154

RESUMO

Encapsulation of charged proteins into complex coacervate core micelles (C3Ms) can be accomplished by mixing them with oppositely charged diblock copolymers. However, these micelles tend to disintegrate at high ionic strength. Previous research showed that the addition of a homopolymer with the same charge sign as the protein improved the stability of protein-containing C3Ms. In this research, we used fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to study how the addition of the homopolymer affects the encapsulation efficiency and salt stability of the micelles. We studied the encapsulation of laccase spore coat protein A (CotA), a multicopper oxidase, using a strong cationic-neutral diblock copolymer, poly(N-methyl-2-vinyl-pyridinium iodide)-block-poly(ethylene oxide) (PM2VP128-b-PEO477), and a negatively charged homopolymer, poly(4-styrenesulfonate) (PSS215). DLS indeed showed an improved stability of this three-component C3M system against the addition of salt compared to a two-component system. Remarkably, FCS showed that the release of CotA from a three-component C3M system occurred at a lower salt concentration and over a narrower concentration range than the dissociation of C3Ms. In conclusion, although the addition of the homopolymer to the system leads to micelles with a higher salt stability, CotA is excluded from the C3Ms already at lower ionic strengths because the homopolymer acts as a competitor of the enzyme for encapsulation.


Assuntos
Micelas , Polietilenoglicóis , Cátions , Polímeros , Espectrometria de Fluorescência
6.
Langmuir ; 34(40): 12083-12092, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30212214

RESUMO

The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128- b-poly(ethylene-oxide)477. Förster resonance energy transfer (FRET) allowed us to determine the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength ( I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of experimental and theoretical considerations, we propose that C3Ms relax to their final state by association and dissociation of near-neutral soluble protein-polymer complexes. To obtain protein-containing C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.


Assuntos
Proteínas de Fluorescência Verde/química , Micelas , Polietilenoglicóis/química , Polivinil/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Químicos , Cloreto de Sódio/química , Termodinâmica
7.
Phys Chem Chem Phys ; 20(10): 7059-7072, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29473921

RESUMO

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.


Assuntos
Apoproteínas/química , Flavodoxina/química , Dobramento de Proteína , Azotobacter vinelandii/química , Sítios de Ligação , Cinética , Modelos Moleculares , Concentração Osmolar , Ligação Proteica , Estrutura Secundária de Proteína , Termodinâmica , Triptofano/química
8.
Appl Microbiol Biotechnol ; 102(3): 1281-1295, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29196788

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a ß-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.


Assuntos
Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Biocombustíveis , Biomassa , Catálise , Quitina/metabolismo , Cobre/metabolismo , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Oligossacarídeos/metabolismo , Oxirredução , Plantas/química , Sordariales/enzimologia , Temperatura , beta-Glucosidase/metabolismo
9.
J Biol Chem ; 291(50): 25911-25920, 2016 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-27784783

RESUMO

Folding of proteins usually involves intermediates, of which an important type is the molten globule (MG). MGs are ensembles of interconverting conformers that contain (non-)native secondary structure and lack the tightly packed tertiary structure of natively folded globular proteins. Whereas MGs of various purified proteins have been probed to date, no data are available on their presence and/or effect during protein synthesis. To study whether MGs arise during translation, we use ribosome-nascent chain (RNC) complexes of the electron transfer protein flavodoxin. Full-length isolated flavodoxin, which contains a non-covalently bound flavin mononucleotide (FMN) as cofactor, acquires its native α/ß parallel topology via a folding mechanism that contains an off-pathway intermediate with molten globular characteristics. Extensive population of this MG state occurs at physiological ionic strength for apoflavodoxin variant F44Y, in which a phenylalanine at position 44 is changed to a tyrosine. Here, we show for the first time that ascertaining the binding rate of FMN as a function of ionic strength can be used as a tool to determine the presence of the off-pathway MG on the ribosome. Application of this methodology to F44Y apoflavodoxin RNCs shows that at physiological ionic strength the ribosome influences formation of the off-pathway MG and forces the nascent chain toward the native state.


Assuntos
Azotobacter vinelandii/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavodoxina/biossíntese , Dobramento de Proteína , Ribossomos/metabolismo , Substituição de Aminoácidos , Azotobacter vinelandii/genética , Mononucleotídeo de Flavina/genética , Flavodoxina/genética , Mutação de Sentido Incorreto , Ribossomos/genética
10.
Biochim Biophys Acta Proteins Proteom ; 1865(12): 1770-1780, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28888693

RESUMO

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s-1, one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity.


Assuntos
Oxigenases/metabolismo , Catálise , Mononucleotídeo de Flavina/farmacologia , Modelos Moleculares , Oxigenases/química , Oxigenases/genética , Rhodococcus/enzimologia , Alinhamento de Sequência
11.
Bioconjug Chem ; 28(4): 1189-1193, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28263569

RESUMO

Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidation of tyrosine with mushroom tyrosinase, strain-promoted cycloaddition (SPOCQ) of the resulting 1,2-quinone with various bicyclo[6.1.0]nonyne (BCN) derivatives led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific preparation of an antibody-drug conjugate.


Assuntos
Imunoconjugados/química , Monofenol Mono-Oxigenase/química , Coloração e Rotulagem/métodos , Tirosina/química , Celulases , Reação de Cicloadição , Oxirredução
12.
Int J Mol Sci ; 18(7)2017 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-28753915

RESUMO

Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry).


Assuntos
Antozoários/metabolismo , Proteínas de Fluorescência Verde/química , Polietilenoglicóis/química , Animais , Sistemas de Liberação de Medicamentos , Difusão Dinâmica da Luz , Concentração de Íons de Hidrogênio , Micelas , Modelos Moleculares , Teoria Quântica , Espectrometria de Fluorescência
13.
Biochim Biophys Acta ; 1854(10 Pt A): 1317-24, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26073784

RESUMO

Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/ß parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN.


Assuntos
Apoproteínas/química , Azotobacter vinelandii/química , Proteínas de Bactérias/química , Mononucleotídeo de Flavina/química , Flavodoxina/química , Ribossomos/química , Apoproteínas/genética , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Flavodoxina/genética , Expressão Gênica , Modelos Moleculares , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ribossomos/metabolismo
14.
Fish Shellfish Immunol ; 56: 70-83, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27368535

RESUMO

Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.


Assuntos
Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Sequência de Aminoácidos , Animais , Carpas/classificação , Carpas/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Filogenia , Alinhamento de Sequência , Sintenia , Receptor 1 Toll-Like/química , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/química , Receptor 2 Toll-Like/imunologia
15.
Biomacromolecules ; 16(5): 1542-9, 2015 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-25857527

RESUMO

Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms.


Assuntos
Sistemas de Liberação de Medicamentos , Proteínas de Fluorescência Verde/química , Polímeros/química , Humanos , Concentração de Íons de Hidrogênio , Micelas , Polietilenoglicóis/química
16.
Proc Natl Acad Sci U S A ; 109(5): 1560-5, 2012 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-22238427

RESUMO

Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Flores , Proteínas de Domínio MADS/metabolismo , Arabidopsis/metabolismo , Cromatografia de Afinidade , Espectrometria de Massas
17.
J Biol Chem ; 288(36): 26235-26245, 2013 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-23864660

RESUMO

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.


Assuntos
Proteínas de Bactérias/química , Flavoproteínas/química , Oxigenases de Função Mista/química , Fosfolipídeos/química , Rhodococcus/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Expressão Gênica , Gentisatos/química , Gentisatos/metabolismo , Hidroxilação , Espectrometria de Massas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mutação de Sentido Incorreto , NAD/química , NAD/genética , NAD/metabolismo , Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/genética
18.
J Gen Virol ; 95(Pt 2): 245-262, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24225498

RESUMO

Many viruses have evolved strategies to deregulate the host immune system. These strategies include mechanisms to subvert or recruit the host cytokine network. IL-10 is a pleiotropic cytokine that has both immunostimulatory and immunosuppressive properties. However, its key features relate mainly to its capacity to exert potent immunosuppressive effects. Several viruses have been shown to upregulate the expression of cellular IL-10 (cIL-10) with, in some cases, enhancement of infection by suppression of immune functions. Other viruses encode functional orthologues of cIL-10, called viral IL-10s (vIL-10s). The present review is devoted to these virokines. To date, vIL-10 orthologues have been reported for 12 members of the family Herpesviridae, two members of the family Alloherpesviridae and seven members of the family Poxviridae. Study of vIL-10s demonstrated several interesting aspects on the origin and the evolution of these viral genes, e.g. the existence of multiple (potentially up to nine) independent gene acquisition events at different times during evolution, viral gene acquisition resulting from recombination with cellular genomic DNA or cDNA derived from cellular mRNA and the evolution of cellular sequence in the viral genome to restrict the biological activities of the viral orthologues to those beneficial for the virus life cycle. Here, various aspects of the vIL-10s described to date are reviewed, including their genetic organization, protein structure, origin, evolution, biological properties and potential in applied research.


Assuntos
Herpesviridae/fisiologia , Evasão da Resposta Imune , Tolerância Imunológica , Interleucina-10/metabolismo , Poxviridae/fisiologia , Proteínas Virais/metabolismo , Fatores de Virulência/metabolismo , Animais , Evolução Molecular , Herpesviridae/genética , Humanos , Interleucina-10/genética , Poxviridae/genética , Proteínas Virais/genética , Fatores de Virulência/genética
19.
Plant Physiol ; 162(4): 1911-25, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23796795

RESUMO

The leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) is the main ligand-perceiving receptor for brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana). Binding of BRs to the ectodomain of plasma membrane (PM)-located BRI1 receptors initiates an intracellular signal transduction cascade that influences various aspects of plant growth and development. Even though the major components of BR signaling have been revealed and the PM was identified as the main site of BRI1 signaling activity, the very first steps of signal transmission are still elusive. Recently, it was shown that the initiation of BR signal transduction requires the interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors. In addition, the resolved structure of the BRI1 ectodomain suggested that BRI1-ASSOCIATED KINASE1 [BAK1](SERK3) may constitute a component of the ligand-perceiving receptor complex. Therefore, we investigated the spatial correlation between BRI1 and BAK1(SERK3) in the natural habitat of both leucine-rich repeat receptor-like kinases using comparative colocalization analysis and fluorescence lifetime imaging microscopy. We show that activation of BR signaling by exogenous ligand application resulted in both elevated colocalization between BRI1 and BAK1(SERK3) and an about 50% increase of receptor heterooligomerization in the PM of live Arabidopsis root epidermal cells. However, large populations of BRI1 and BAK1(SERK3) colocalized independently of BRs. Moreover, we could visualize that approximately 7% of the BRI1 PM pool constitutively heterooligomerizes with BAK1(SERK3) in live root cells. We propose that only small populations of PM-located BRI1 and BAK1(SERK3) receptors participate in active BR signaling and that the initiation of downstream signal transduction involves preassembled BRI1-BAK1(SERK3) heterooligomers.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brefeldina A/metabolismo , Brefeldina A/farmacologia , Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Triazóis/farmacologia
20.
Arch Biochem Biophys ; 544: 2-17, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24361254

RESUMO

Flavin-dependent monooxygenases catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. As such, they are involved in key biological processes ranging from catabolism, detoxification and biosynthesis, to light emission and axon guidance. Based on fold and function, flavin-dependent monooxygenases can be distributed into eight groups. Groups A and B comprise enzymes that rely on NAD(P)H as external electron donor. Groups C-F are two-protein systems, composed of a monooxygenase and a flavin reductase. Groups G and H comprise internal monooxygenases that reduce the flavin cofactor through substrate oxidation. Recently, many new flavin-dependent monooxygenases have been discovered. In addition to posing basic enzymological questions, these proteins attract attention of pharmaceutical and fine-chemical industries, given their importance as regio- and enantioselective biocatalysts. In this review we present an update of the classification of flavin-dependent monooxygenases and summarize the latest advances in our understanding of their catalytic and structural properties.


Assuntos
Flavinas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Animais , Humanos , Modelos Moleculares , Oxirredutases/química , Oxirredutases/metabolismo , Conformação Proteica
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