RESUMEN
Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , Akkermansia , Glicósido Hidrolasas , Sistema del Grupo Sanguíneo ABO/inmunología , Humanos , Glicósido Hidrolasas/metabolismo , Mucinas/metabolismo , Eritrocitos/inmunología , Polisacáridos/metabolismo , Microbioma Gastrointestinal , Antígenos de Grupos Sanguíneos/metabolismo , Antígenos de Grupos Sanguíneos/inmunología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/inmunologíaRESUMEN
We report here the identification, characterization and three-dimensional (3D) structure determination of NaNga, a newly identified ß-N-acetylgalactosaminidase from the Gram-negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4-nitrophenyl-N-acetyl-ß-d-galactosamine (pNP-ß-d-GalpNAc). The X-ray crystal structure of the protein was refined to 2.5 Å and consists of an N-terminal ß-sandwich domain and a (ß/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate-assisted catalytic mechanism. Inhibition by N-acetyl-galactosamine thiazoline (GalNAc-thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a ß-N-acetylgalactosaminide substituted by a monosaccharide at O-3. Analysis of the genomic context suggests, in turn, that this substituted ß-N-acetylgalactosaminide may be appended to a d-arabinan from an environmental Actinomycete.
Asunto(s)
Bacteroidetes , Galactosamina , beta-N-Acetil-Galactosaminidasa , Filogenia , Dominio Catalítico , Especificidad por SustratoRESUMEN
Fucoidanases (EC 3.2.1.-) catalyze the hydrolysis of glycosidic bonds between fucose residues in fucoidans. Fucoidans are a compositionally and structurally diverse class of fucose-containing sulfated polysaccharides that are primarily found in brown seaweeds. Here, the structural characterization of a novel endo-α(1,4)-fucoidanase, Mef1, from the marine bacterium Muricauda eckloniae is presented, showing sequence similarity to members of glycoside hydrolase family 107. Using carbohydrate polyacrylamide gel electrophoresis and nuclear magnetic resonance analyses, it is shown that the fucoidanase Mef1 catalyzes the cleavage of α(1,4)-linkages between fucose residues sulfated on C2 in the structure [-3)-α-L-Fucp2S-(1,4)-α-L-Fucp2S-(1-]n in fucoidan from Fucus evanescens. Kinetic analysis of Mef1 activity by Fourier transform infrared spectroscopy revealed that the specific Mef1 fucoidanase activity (Uf) on F. evanescens fucoidan was 0.1 × 10-3â Ufâ µM-1. By crystal structure determination of Mef1 at 1.8â Å resolution, a single-domain organization comprising a (ß/α)8-barrel domain was determined. The active site was in an extended, positively charged groove that is likely to be designed to accommodate the binding of the negatively charged, sulfated fucoidan substrate. The active site of Mef1 comprises the amino acids His270 and Asp187, providing acid/base and nucleophile groups, respectively, for the hydrolysis of glycosidic bonds in the fucoidan backbone. Electron densities were identified for two possible Ca2+ ions in the enzyme, one of which is partially exposed to the active-site groove, while the other is very tightly coordinated. A water wire was discovered leading from the exterior of the Mef1 enzyme into the active site, passing the tightly coordinated Ca2+ site.
Asunto(s)
Flavobacteriaceae , Fucosa , Cinética , Polisacáridos/química , Glicósido Hidrolasas/química , Flavobacteriaceae/metabolismoRESUMEN
Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.
Asunto(s)
Conotoxinas , Ratones , Animales , Conotoxinas/farmacología , Conotoxinas/química , Canales de Calcio , Péptidos/química , Células Receptoras Sensoriales/metabolismo , CaracolesRESUMEN
Marine cone snails produce a wealth of peptide toxins (conotoxins) that bind their molecular targets with high selectivity and potency. Therefore, conotoxins constitute valuable biomolecular tools with a variety of biomedical purposes. The Mu8.1 conotoxin from Conus mucronatus is the founding member of the newly identified saposin-like conotoxin class of conotoxins and has been shown to target Cav2.3, a voltage-gated calcium channel. Two crystal structures have recently been determined of Mu8.1 at 2.3 and 2.1â Å resolution. Here, a high-resolution crystal structure of Mu8.1 was determined at 1.67â Å resolution in the high-symmetry space group I4122. The asymmetric unit contained one molecule, with a symmetry-related molecule generating a dimer equivalent to that observed in the two previously determined structures. The high resolution allows a detailed atomic analysis of a water-filled cavity buried at the dimer interface, revealing a tightly coordinated network of waters that shield a lysine residue (Lys55) with a predicted unusually low side-chain pKa value. These findings are discussed in terms of a potential functional role of Lys55 in target interaction.
Asunto(s)
Conotoxinas , Caracol Conus , Animales , Cristalografía por Rayos X , Lisina , AguaRESUMEN
Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.
Asunto(s)
Histidina , Oxigenasas de Función Mixta , Oxigenasas de Función Mixta/metabolismo , Histidina/genética , Histidina/metabolismo , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Polisacáridos/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.
Asunto(s)
Mucinas , Neuraminidasa , Humanos , Mucinas/metabolismo , Neuraminidasa/metabolismo , alfa-L-Fucosidasa/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fucosa/metabolismo , Verrucomicrobia/metabolismo , Polisacáridos/metabolismo , Moco/metabolismoRESUMEN
BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Insuficiencia Cardíaca , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Humanos , Ratones , Calcio/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3/metabolismo , Insuficiencia Cardíaca/metabolismo , Células HEK293 , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
We apply a computational model, global multi-mutant analysis (GMMA), to inform on effects of most amino acid substitutions from a randomly mutated gene library. Using a high mutation frequency, the method can determine mutations that increase the stability of even very stable proteins for which conventional selection systems have reached their limit. As a demonstration of this, we screened a mutant library of a highly stable and computationally redesigned model protein using an in vivo genetic sensor for folding and assigned a stability effect to 374 of 912 possible single amino acid substitutions. Combining the top 9 substitutions increased the unfolding energy 47 to 69 kJ/mol in a single engineering step. Crystal structures of stabilized variants showed small perturbations in helices 1 and 2, which rendered them closer in structure to the redesign template. This case study illustrates the capability of the method, which is applicable to any screen for protein function.
Asunto(s)
Pliegue de Proteína , Proteínas , Proteínas/genética , Estabilidad Proteica , Mutación , Sustitución de AminoácidosRESUMEN
Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the ß-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65â Å resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.
Asunto(s)
Protones , Xilanos , Catálisis , Dimerización , Glucuronatos/metabolismo , Glicósido Hidrolasas/química , Cinética , Especificidad por Sustrato , Xilanos/metabolismoRESUMEN
Background Mutations in ATP1A2 gene encoding the Na,K-ATPase α2 isoform are associated with familial hemiplegic migraine type 2. Migraine with aura is a known risk factor for heart disease. The Na,K-ATPase is important for cardiac function, but its role for heart disease remains unknown. We hypothesized that ATP1A2 is a susceptibility gene for heart disease and aimed to assess the underlying disease mechanism. Methods and Results Mice heterozygous for the familial hemiplegic migraine type 2-associated G301R mutation in the Atp1a2 gene (α2+/G301R mice) and matching wild-type controls were compared. Reduced expression of the Na,K-ATPase α2 isoform and increased expression of the α1 isoform were observed in hearts from α2+/G301R mice (Western blot). Left ventricular dilation and reduced ejection fraction were shown in hearts from 8-month-old α2+/G301R mice (cardiac magnetic resonance imaging), and this was associated with reduced nocturnal blood pressure (radiotelemetry). Cardiac function and blood pressure of 3-month-old α2+/G301R mice were similar to wild-type mice. Amplified Na,K-ATPase-dependent Src kinase/Ras/Erk1/2 (p44/42 mitogen-activated protein kinase) signaling was observed in hearts from 8-month-old α2+/G301R mice, and this was associated with mitochondrial uncoupling (respirometry), increased oxidative stress (malondialdehyde measurements), and a heart failure-associated metabolic shift (hyperpolarized magnetic resonance). Mitochondrial membrane potential (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolocarbocyanine iodide dye assay) and mitochondrial ultrastructure (transmission electron microscopy) were similar between the groups. Proteomics of heart tissue further suggested amplified Src/Ras/Erk1/2 signaling and increased oxidative stress and provided the molecular basis for systolic dysfunction in 8-month-old α2+/G301R mice. Conclusions Our findings suggest that ATP1A2 mutation leads to disturbed cardiac metabolism and reduced cardiac function mediated via Na,K-ATPase-dependent reactive oxygen species signaling through the Src/Ras/Erk1/2 pathway.
Asunto(s)
Corazón , Trastornos Migrañosos , Migraña con Aura , ATPasa Intercambiadora de Sodio-Potasio , Animales , Corazón/fisiopatología , Heterocigoto , Ratones , Migraña con Aura/metabolismo , Mutación , Miocardio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.
Asunto(s)
Aves , Ojo/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Animales , Humor Acuoso/metabolismo , Ácido Ascórbico/metabolismo , Aves/metabolismo , Ojo/metabolismo , Vuelo AnimalRESUMEN
Magnesium transporter A (MgtA) is an active transporter responsible for importing magnesium ions into the cytoplasm of prokaryotic cells. This study focuses on the peptide corresponding to the intrinsically disordered N-terminal region of MgtA, referred to as KEIF. Primary-structure and bioinformatic analyses were performed, followed by studies of the undisturbed single chain using a combination of techniques including small-angle X-ray scattering, circular dichroism spectroscopy, and atomistic molecular-dynamics simulations. Moreover, interactions with large unilamellar vesicles were investigated by using dynamic light scattering, laser Doppler velocimetry, cryogenic transmission electron microscopy, and circular dichroism spectroscopy. KEIF was confirmed to be intrinsically disordered in aqueous solution, although extended and containing little ß -structure and possibly PPII structure. An increase of helical content was observed in organic solvent, and a similar effect was also seen in aqueous solution containing anionic vesicles. Interactions of cationic KEIF with anionic vesicles led to the hypothesis that KEIF adsorbs to the vesicle surface through electrostatic and entropic driving forces. Considering this, there is a possibility that the biological role of KEIF is to anchor MgtA in the cell membrane, although further investigation is needed to confirm this hypothesis.
Asunto(s)
Adenosina Trifosfatasas/química , Fenómenos Químicos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas de Transporte de Membrana/química , Adenosina Trifosfatasas/ultraestructura , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dicroismo Circular , Proteínas de Escherichia coli/ultraestructura , Proteínas Intrínsecamente Desordenadas/ultraestructura , Lípidos/química , Proteínas de Transporte de Membrana/ultraestructura , Simulación de Dinámica Molecular , Tamaño de la Partícula , Probabilidad , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Liposomas Unilamelares/química , Difracción de Rayos XRESUMEN
Thermostable cellulases from glycoside hydrolase family 7 (GH7) are the main components of enzymatic mixtures for industrial saccharification of lignocellulose. Activity improvement of these enzymes via rational design is a promising strategy to alleviate the industrial costs, but it requires detailed structural knowledge. While substantial biochemical and structural data are available for GH7 cellobiohydrolases, endoglucanases are more elusive and only few structures have been solved so far. Here, we report a new crystal structure and biochemical characterization of a thermostable endoglucanase from the thermophilic ascomycete Rasamsonia emersonii, ReCel7B. The enzyme was compared with the homologous endoglucanase from the mesophilic model ascomycete Trichoderma reesei (TrCel7B), which unlike ReCel7B possesses an additional carbohydrate-binding module (CBM). With a temperature optimum of 80 °C, ReCel7B displayed a number of differences in activity and ability to synergize with cellobiohydrolases compared to TrCel7B. We improved both binding and kinetics in a chimeric variant of ReCel7B and a CBM, while we observe the opposite effect when the CBM was removed in TrCel7B. The crystal structure of ReCel7B was determined at 2.48 Å resolution, with Rwork and Rfree factors of 0.182 and 0.206, respectively. Structural analyses revealed that ReCel7B has increased rigidity in a number of peripheral loops compared to TrCel7B and fewer aromatics in the substrate-binding cleft. An increased number of glycosylations were identified in ReCel7B, and we propose a stabilizing mechanism for one of the glycans. Global structure-function interpretations of ReCel7B highlight the differences in temperature stability, turnover, binding, and cellulose accessibility in GH7 endoglucanases. DATABASE: Structural data are available in RCSB Protein Data Bank database under the accession number 6SU8. ENZYMES: ReCel7B, endoglucanase (EC3.2.1.4) from Rasamsonia emersonii; ReCel7A, cellobiohydrolase (EC3.2.1.176) from Rasamsonia emersonii; TrCel7B, endoglucanase (EC3.2.1.4) from Trichoderma reesei; TrCel7A, cellobiohydrolase (EC3.2.1.176) from Trichoderma reesei.
Asunto(s)
Celulasa/química , Celulasa/metabolismo , Eurotiales/enzimología , Temperatura , Cinética , Modelos Moleculares , Filogenia , Conformación ProteicaRESUMEN
Factor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.
Asunto(s)
Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Animales , Biocatálisis/efectos de los fármacos , Calcio/farmacología , Dominio Catalítico , Iones , Cinética , Sustancias Macromoleculares/metabolismo , Enfermedad del Virus de Marburg/enzimología , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dominios Proteicos , Pliegue de Proteína/efectos de los fármacos , Sodio/farmacología , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacosRESUMEN
Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro, which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Endosomas/enzimología , Endosomas/genética , Proteínas Activadoras de GTPasa/genética , Aparato de Golgi/enzimología , Aparato de Golgi/genética , Humanos , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
We performed quantum-chemical calculations, ab initio molecular dynamics, hybrid quantum mechanics/molecular mechanics (QM/MM) and enhanced sampling metadynamics simulations to investigate the origin of metal specificity in isatin hydrolase from Labrenzia aggregata. The peculiar octahedral binding geometry of the Mn2+ ion in the Michaelis complex includes both the isatin substrate and the catalytic water within the first coordination shell of the cation. Our calculations show that the same arrangement of the ligands cannot be efficiently achieved in the presence of other small divalent metal cations such as Zn2+ or Cu2+ . On the contrary, bulkier alkaline-earth cations such as Mg2+ , which allow octahedral coordination, are not able to activate the catalytic water into the stronger OH- nucleophile required to attack the stable N-aryl-amide moiety of isatin.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Isatina/química , Metales/química , Simulación de Dinámica Molecular , Rhodobacteraceae/enzimología , Cationes Bivalentes/química , Simulación por Computador , Ligandos , Unión Proteica , Teoría Cuántica , Termodinámica , Agua/químicaRESUMEN
Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.
Asunto(s)
Dominio Catalítico/genética , Deficiencia del Factor VII/genética , Factor VII/química , Factor VII/genética , Mutación Missense , Sustitución de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/metabolismo , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Pliegue de Proteína , Transporte de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
A-kinase anchoring proteins (AKAPs) are a family of proteins that provide spatiotemporal resolution of protein kinase A (PKA) phosphorylation. In the myocardium, PKA and AKAP18γ/δ are found in complex with sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and phospholamban (PLB). This macromolecular complex provides a means by which anchored PKA can dynamically regulate cytoplasmic Ca(2+) release and re-uptake. For this reason, AKAP18γ/δ presents an interesting drug target with therapeutic potential in cardiovascular disease. The crystal structure of the central domain of human AKAP18γ has been determined at the atomic resolution of 1.25â Å. This first structure of human AKAP18γ is trapped in a novel conformation by a malonate molecule bridging the important R-loop with the 2H phosphoesterase motif. Although the physiological substrate of AKAP18γ is currently unknown, a potential proton wire deep in the central binding crevice has been indentified, leading to bulk solvent below the R-loop. Malonate complexed with AKAP18γ at atomic resolution provides an excellent starting point for structure-guided drug design.