RESUMEN

The three-dimensional structure of proteins determines their function in vital biological processes. Thus, when the structure is known, the molecular mechanism of protein function can be understood in more detail and obtained information utilized in biotechnological, diagnostics, and therapeutic applications. Over the past five years, machine learning (ML)-based modeling has pushed protein structure prediction to the next level with AlphaFold in the front line, predicting the structure for hundreds of millions of proteins. Further advances recently report promising ML-based approaches for solving remaining challenges by incorporating functionally important metals, co-factors, post-translational modifications, structural dynamics, and interdomain and multimer interactions in the structure prediction process.

Asunto(s)

Aprendizaje Automático , Conformación Proteica , Proteínas , Proteínas/química , Proteínas/metabolismo , Modelos Moleculares , Biología Computacional/métodos

RESUMEN

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.

Asunto(s)

Integrina alfa5beta1 , Glándulas Sebáceas , Animales , Adhesión Celular , Diferenciación Celular , Fibronectinas , Integrina beta1 , Integrinas/metabolismo , Ratones

RESUMEN

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.

Asunto(s)

Neoplasias de la Mama/patología , Carcinogénesis , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Receptor Notch3/metabolismo , Transporte Activo de Núcleo Celular , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones , Modelos Moleculares , Proteínas Musculares/metabolismo , Fosforilación , Dominios Proteicos , Receptor Notch3/química

RESUMEN

The co-chaperone CHIP (carboxy terminus of Hsc70 interacting protein) is very important for many cell activities since it regulates the ubiquitination of substrates targeted for proteasomal degradation. However, information on the structure-function relationship of CHIP from plants and how it interacts and ubiquitinates other plant chaperones is still needed. For that, the CHIP ortholog from Sorghum bicolor (SbCHIP) was identified and studied in detail. SbCHIP was purified and produced folded and pure, being capable of keeping its structural conformation up to 42 °C, indicating that cellular function is maintained even in a hot environment. Also, SbCHIP was able to bind plant Hsp70 and Hsp90 with high affinity and interact with E2 enzymes, performing E3 ligase activity. The data allowed to reveal the pattern of plant Hsp70 and Hsp90 ubiquitination and described which plant E2 enzymes are likely involved in SbCHIP-mediated ubiquitination. Aditionally, we obtained information on the SbCHIP conformation, showing that it is a non-globular symmetric dimer and allowing to put forward a model for the interaction of SbCHIP with chaperones and E2 enzymes that suggests a mechanism of ubiquitination. Altogether, the results presented here are useful additions to the study of protein folding and degradation in plants.

Asunto(s)

Proteínas del Choque Térmico HSC70/metabolismo , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sorghum/metabolismo , Dicroismo Circular , Filogenia , Proteínas de Plantas/genética , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia , Sorghum/genética , Resonancia por Plasmón de Superficie , Ubiquitinación , Difracción de Rayos X

RESUMEN

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.

Asunto(s)

Amina Oxidasa (conteniendo Cobre)/química , Moléculas de Adhesión Celular/genética , Diseño de Fármacos , Desarrollo de Medicamentos/tendencias , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/uso terapéutico , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/uso terapéutico , Histamina/química , Humanos

RESUMEN

The enantiomers of aromatic 4-dibenzocyclooctynol (DIBO), used for radiolabeling and subsequent conjugation of biomolecules to form radioligands for positron emission tomography (PET), were separated by kinetic resolution using lipase A from Candida antarctica (CAL-A). In optimized conditions, (R)-DIBO [(R)-1, ee 95%] and its acetylated (S)-ester [(S)-2, ee 96%] were isolated. In silico docking results explained the ability of CAL-A to differentiate the enantiomers of DIBO and to accommodate various acyl donors. Anhydrous MgCl2 was used for binding water from the reaction medium and, thus, for obtaining higher conversion by preventing hydrolysis of the product (S)-2 into the starting material. Since the presence of hydrated MgCl26H2O also allowed high conversion or effect on enantioselectivity, Mg2+ ion was suspected to interact with the enzyme. Binding site predictions indicated at least two sites of interest; one in the lid domain at the bottom of the acyl binding pocket and another at the interface of the hydrolase and flap domains, just above the active site.

Asunto(s)

Candida/enzimología , Lipasa/metabolismo , Tomografía de Emisión de Positrones , Sitios de Unión , Biocatálisis , Dominio Catalítico , Desecación , Esterificación , Iones , Cinética , Magnesio/farmacología , Conformación Molecular , Simulación del Acoplamiento Molecular , Estereoisomerismo

RESUMEN

Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated.

Asunto(s)

Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sorghum/química , Productos Agrícolas , Proteínas de Choque Térmico/química , Humanos , Conformación Molecular , Proteínas de Plantas/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química

RESUMEN

The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.

Asunto(s)

Regulación de la Expresión Génica , Receptor Notch1/química , Receptor Notch1/metabolismo , Estrés Fisiológico , Sumoilación , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Estrés Oxidativo , Transducción de Señal

RESUMEN

Plastidic ferredoxin-NADP+ oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP+ oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.2 ± 0.2 s-1 , KmNADPH = 1.6 ± 0.3 µM and kcat /KmNADPH = 2.0 ± 0.4 µM-1 s-1 . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded ß-sheet and one α-helix, and a C-terminal NADP+ -binding α/ß domain with a five-stranded ß-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas de Cloroplastos/metabolismo , Ferredoxina-NADP Reductasa/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Proteínas de Cloroplastos/química , Proteínas de Cloroplastos/genética , Ferredoxina-NADP Reductasa/clasificación , Ferredoxina-NADP Reductasa/genética , Flavina-Adenina Dinucleótido/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Cinética , Modelos Moleculares , Filogenia , Dominios Proteicos , Homología de Secuencia de Aminoácido

RESUMEN

The universally conserved TsaC/TsaC2/YciO family of proteins is essential for the N6-threonylcarbamoyladenosine modification present in almost all ANN-decoding tRNAs. Previously, the family has been grouped into the TsaC/TsaC2 and YciO subfamilies. We used sequence analysis, phylogenetic methods and homology modeling to show that a third subfamily, the Slr0006-like subfamily, exists exclusively in some cyanobacteria. The Slr0006-like proteins are solely found together with both TsaC and YciO homologs, indicating a distinct function for the Slr0006-like subfamily. Accordingly, the homology models show that the amino acids in their putative binding clefts differ significantly. Hence, we introduce a new cyanobacterial subfamily of proteins with the TsaC-domain fold, along with the generated classification rules to assign new members to the correct cyanobacterial subfamily.

Asunto(s)

Proteínas Bacterianas/química , Synechocystis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Evolución Molecular , Simulación de Dinámica Molecular , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Filogenia , Dominios Proteicos , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína

RESUMEN

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Asunto(s)

Infecciones por Klebsiella/metabolismo , Klebsiella pneumoniae/metabolismo , Lípido A/biosíntesis , Lípido A/química , Animales , Humanos , Infecciones por Klebsiella/genética , Infecciones por Klebsiella/patología , Klebsiella pneumoniae/genética , Lípido A/genética , Pulmón/microbiología , Ratones , Estructura Molecular , Especificidad de Órganos

RESUMEN

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A.

Asunto(s)

Autoantígenos/química , Proteínas de la Membrana/química , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Relación Estructura-Actividad Cuantitativa , Alineación de Secuencia , Electricidad Estática

RESUMEN

Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants' survival in a fluctuating environment. These changes may be achieved through posttranslational modifications of proteins, which are known to affect the activity, interactions, and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation, and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type FERREDOXIN-NADP(+) OXIDOREDUCTASE (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both AtFNR isoforms contain multiple alternative amino termini and undergo light-responsive addition of an acetyl group to the α-amino group of the amino-terminal amino acid of proteins, which causes the change in isoelectric point. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of AtFNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of posttranslational modifications, which continues to emerge as a novel area within photosynthesis research.

Asunto(s)

Arabidopsis/enzimología , Ferredoxina-NADP Reductasa/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimología , Ferredoxina-NADP Reductasa/genética , Ferredoxinas/metabolismo , Glicosilación , Isoenzimas , Luz , Modelos Estructurales , Datos de Secuencia Molecular , NADP/metabolismo , Fosforilación , Fotosíntesis , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Alineación de Secuencia

RESUMEN

We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via π-π interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

Asunto(s)

Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Evolución Molecular Dirigida , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas de Alcohol/química , Sustitución de Aminoácidos , Dominio Catalítico , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Especificidad por Sustrato , Termodinámica

RESUMEN

The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments.

Asunto(s)

Bryopsida/metabolismo , Proteínas Portadoras/metabolismo , Frío , Sequías , Ácidos Grasos Insaturados/metabolismo , Proteínas Ligadas a GPI/metabolismo , Proteínas de Plantas/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Bryopsida/química , Proteínas Portadoras/química , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/química , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos de la Membrana/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Unión Proteica , Estrés Fisiológico , Regulación hacia Arriba

RESUMEN

Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.

RESUMEN

The slr0006 gene of Synechocystis sp. PCC 6803 is upregulated at mRNA and protein level under carbon limitation. The T(N11)A motif in the upstream region of slr0006 is a binding site for transcriptional regulator NdhR, and accumulation of the Slr0006 protein in ndhR deletion mutant grown in high CO2 suggests that NdhR may be a negative regulator of slr0006. Accumulation requires photosynthetic electron transfer, because no Slr0006 was detected in darkness or in the presence of electron transfer inhibitors DCMU and DBMIB. Structural modeling of the Slr0006 protein suggests that it adopts Sua5/YciO/YrdC family fold, which is an α/ß twisted open-sheet structure. Similar to the structurally known members of this protein family, the surface of Slr0006 contains positively charged cavity indicating a possible binding site for RNA or nucleotides. Moreover, Slr0006 was co-localized with 30S ribosomal proteins and rRNA, suggesting involvement in processes linked to protein synthesis.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biosíntesis de Proteínas , Synechocystis/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Dióxido de Carbono/metabolismo , Oscuridad , Transporte de Electrón , Regulación de la Expresión Génica , Glicosiltransferasas/química , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Datos de Secuencia Molecular , Fotosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Synechocystis/genética , Synechocystis/metabolismo , Regulación hacia Arriba

RESUMEN

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

Asunto(s)

Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Lípido A/química , Lípido A/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidad , Acilación , Adhesinas Bacterianas/biosíntesis , Animales , Arabinosa/análogos & derivados , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Mutación , Ácidos Palmíticos , Temperatura , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Yersiniosis/genética , Yersiniosis/inmunología , Yersiniosis/microbiología , Yersinia enterocolitica/genética , Yersinia enterocolitica/inmunología