RESUMEN
The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.
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Oxitocina , Receptores de Oxitocina , Microscopía por Crioelectrón , Humanos , Ligandos , Oxitocina/metabolismo , Elementos Estructurales de las Proteínas , Receptores de Oxitocina/química , Receptores de Oxitocina/genética , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/química , Receptores de Vasopresinas/metabolismo , Relación Estructura-ActividadRESUMEN
The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.
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COVID-19/fisiopatología , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Catepsinas/metabolismo , Receptores ErbB/antagonistas & inhibidores , Humanos , Esquemas de Inmunización , Inmunización Secundaria , Fitoterapia/métodos , Plantas Medicinales , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Elementos Estructurales de las Proteínas/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Inhibidores de Proteasa Viral/farmacología , Inhibidores de Proteasa Viral/uso terapéuticoRESUMEN
Structural bioinformatics suffers from the lack of interfaces connecting biological structures and machine learning methods, making the application of modern neural network architectures impractical. This negatively affects the development of structure-based bioinformatics methods, causing a bottleneck in biological research. Here we present PyUUL ( https://pyuul.readthedocs.io/ ), a library to translate biological structures into 3D tensors, allowing an out-of-the-box application of state-of-the-art deep learning algorithms. The library converts biological macromolecules to data structures typical of computer vision, such as voxels and point clouds, for which extensive machine learning research has been performed. Moreover, PyUUL allows an out-of-the box GPU and sparse calculation. Finally, we demonstrate how PyUUL can be used by researchers to address some typical bioinformatics problems, such as structure recognition and docking.
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Biología Computacional/métodos , Aprendizaje Profundo , Imagenología Tridimensional/métodos , Redes Neurales de la Computación , Algoritmos , Humanos , Elementos Estructurales de las Proteínas/fisiologíaRESUMEN
Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.
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Alphavirus/metabolismo , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Elementos Estructurales de las Proteínas , Replicación ViralRESUMEN
La obtención de información estructural tridimensional de una proteína es de suma importancia en campos tan variados como la bioquímica funcional, las ciencias de materiales o biomédicas. Siendo actualmente la difracción de rayos X de monocristal el estándar de oro para la consecución de este objetivo, la obtención de dicho monocristal sigue siendo un cuello de botella desde el punto de vista práctico, y poco entendido desde el punto de vista teórico. En este artículo se revisa desde la perspectiva estructural de la proteína la forma en que los rayos X permiten obtener la información estructural y las condiciones fisicoquímicas que permiten la formación de un cristal adecuado para estos experimentos.
Obtaining three-dimensional structural information of a protein is of utmost importance in various fields such as functional biochemistry, materials science, or biomedical sciences. Even though single crystal X-ray diffraction is currently the gold standard for this purpose, growing said single crystal is still a bottleneck from a practical viewpoint, and not fully understood from a theoretical point of view. In this article, we review, from a protein structure perspective, the way X-rays provide structural information, and the physicochemical conditions that promote the formation of an adequate crystal for these experiments.
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Difracción de Rayos X/métodos , Proteínas/farmacología , Elementos Estructurales de las Proteínas , Bioquímica , Diseño de Fármacos , AminoácidosRESUMEN
The field of protein structure prediction has recently been revolutionized through the introduction of deep learning. The current state-of-the-art tool AlphaFold2 can predict highly accurate structures; however, it has a prohibitively long inference time for applications that require the folding of hundreds of sequences. The prediction of protein structure annotations, such as amino acid distances, can be achieved at a higher speed with existing tools, such as the ProSPr network. Here, we report on important updates to the ProSPr network, its performance in the recent Critical Assessment of Techniques for Protein Structure Prediction (CASP14) competition, and an evaluation of its accuracy dependency on sequence length and multiple sequence alignment depth. We also provide a detailed description of the architecture and the training process, accompanied by reusable code. This work is anticipated to provide a solid foundation for the further development of protein distance prediction tools.
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Redes Neurales de la Computación , Proteínas/química , Secuencia de Aminoácidos , Biología Computacional/métodos , Humanos , Conformación Proteica , Pliegue de Proteína , Elementos Estructurales de las Proteínas , Alineación de Secuencia/métodos , Diseño de SoftwareRESUMEN
The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid ß aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.
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Chaperonas Moleculares/metabolismo , Enfermedades Neurodegenerativas/prevención & control , Agregación Patológica de Proteínas/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Proteínas tau/metabolismo , Fenómenos Biofísicos , Línea Celular , Humanos , Cinética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica , Elementos Estructurales de las ProteínasRESUMEN
Potts models and variational autoencoders (VAEs) have recently gained popularity as generative protein sequence models (GPSMs) to explore fitness landscapes and predict mutation effects. Despite encouraging results, current model evaluation metrics leave unclear whether GPSMs faithfully reproduce the complex multi-residue mutational patterns observed in natural sequences due to epistasis. Here, we develop a set of sequence statistics to assess the "generative capacity" of three current GPSMs: the pairwise Potts Hamiltonian, the VAE, and the site-independent model. We show that the Potts model's generative capacity is largest, as the higher-order mutational statistics generated by the model agree with those observed for natural sequences, while the VAE's lies between the Potts and site-independent models. Importantly, our work provides a new framework for evaluating and interpreting GPSM accuracy which emphasizes the role of higher-order covariation and epistasis, with broader implications for probabilistic sequence models in general.
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Mutación , Proteínas/química , Alineación de Secuencia/métodos , Algoritmos , Secuencia de Aminoácidos , Simulación por Computador , Bases de Datos de Proteínas , Humanos , Modelos Estadísticos , Elementos Estructurales de las Proteínas , Proteínas/genética , Relación Estructura-ActividadRESUMEN
The Ff family of filamentous bacteriophages infect gram-negative bacteria, but do not cause lysis of their host cell. Instead, new virions are extruded via the phage-encoded pIV protein, which has homology with bacterial secretins. Here, we determine the structure of pIV from the f1 filamentous bacteriophage at 2.7 Å resolution by cryo-electron microscopy, the first near-atomic structure of a phage secretin. Fifteen f1 pIV subunits assemble to form a gated channel in the bacterial outer membrane, with associated soluble domains projecting into the periplasm. We model channel opening and propose a mechanism for phage egress. By single-cell microfluidics experiments, we demonstrate the potential for secretins such as pIV to be used as adjuvants to increase the uptake and efficacy of antibiotics in bacteria. Finally, we compare the f1 pIV structure to its homologues to reveal similarities and differences between phage and bacterial secretins.
Asunto(s)
Microscopía por Crioelectrón/métodos , Inovirus/metabolismo , Secretina/química , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Transporte Biológico , Elementos Estructurales de las Proteínas , Alineación de Secuencia , Proteínas no Estructurales Virales/metabolismoRESUMEN
C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.
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Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Tracto Gastrointestinal/metabolismo , Glicósidos/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/química , Cristalografía por Rayos X , Tracto Gastrointestinal/microbiología , Glicósidos/química , Glicosilación , Filogenia , Elementos Estructurales de las Proteínas , Homología de Secuencia , Especificidad por SustratoRESUMEN
Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.
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Receptor del Péptido 1 Similar al Glucagón/química , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos/química , Péptidos/metabolismo , Microscopía por Crioelectrón/métodos , Receptor del Péptido 1 Similar al Glucagón/agonistas , Dominios Proteicos , Elementos Estructurales de las ProteínasRESUMEN
Zinc homeostasis in mammals is constantly and precisely maintained by sophisticated regulatory proteins. Among them, the Zrt/Irt-like protein (ZIP) regulates the influx of zinc into the cytoplasm. In this work, we have employed all-atom molecular dynamics simulations to investigate the Zn2+ transport mechanism in prokaryotic ZIP obtained from Bordetella bronchiseptica (BbZIP) in a membrane bilayer. Additionally, the structural and dynamical transformations of BbZIP during this process have been analyzed. This study allowed us to develop a hypothesis for the zinc influx mechanism and formation of the metal-binding site. We have created a model for the outward-facing form of BbZIP (experimentally only the inward-facing form has been characterized) that has allowed us, for the first time, to observe the Zn2+ ion entering the channel and binding to the negatively charged M2 site. It is thought that the M2 site is less favored than the M1 site, which then leads to metal ion egress; however, we have not observed the M1 site being occupied in our simulations. Furthermore, removing both Zn2+ ions from this complex resulted in the collapse of the metal-binding site, illustrating the "structural role" of metal ions in maintaining the binding site and holding the proteins together. Finally, due to the long Cd2+-residue bond distances observed in the X-ray structures, we have proposed the existence of an H3O+ ion at the M2 site that plays an important role in protein stability in the absence of the metal ion.
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Bordetella bronchiseptica/metabolismo , Proteínas Portadoras/química , Proteínas de Transporte de Catión/metabolismo , Simulación por Computador/normas , Zinc/metabolismo , Proteínas Portadoras/metabolismo , Simulación de Dinámica Molecular , Elementos Estructurales de las ProteínasRESUMEN
Artificial intelligence technologies such as machine learning have been applied to protein engineering, with unique advantages in protein structure, function prediction, catalytic activity, and other issues in recent years. Screening better mutants is still a bottleneck in protein engineering. In this paper, a new sequence-activity relationship method was analyzed for its application in improving the thermal stability of Aspergillus terreus (R)-ω-selective amine transaminase. The experimental data from 6 single-point mutated enzymes were used as a learning dataset to build models and predict the thermostability of 26 mutants. Based on digital signal processing (DSP), this method digitized the amino acid sequence of proteins by fast Fourier transform (FFT) and then established the best model applying partial least squares regression (PLSR) to screen out all possible mutants, especially those with high performance. In protein engineering, the innovative sequence activity relationship (ISAR) method can make a reasonable prediction using limited experimental data and significantly reduce the experimental cost. The half-life (T 1/2) of (R)-ω-transaminase was fitted with the amino acid sequence by the ISAR algorithm, resulting in an R 2 of 0.8929 and a cvRMSE of 4.89. At the same time, the mutants with higher T 1/2 than the existing ones were predicted, laying the groundwork for better (R)-ω-transaminase in the later stage. The ISAR algorithm is expected to provide a new technique for protein evolution and screening.
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Aminas/química , Aspergillus/enzimología , Aprendizaje Automático , Transaminasas/química , Aminas/metabolismo , Aspergillus/química , Calor , Modelos Moleculares , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Elementos Estructurales de las Proteínas , Especificidad por Sustrato , Transaminasas/metabolismoRESUMEN
tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5'-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5'-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5'-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.
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Proteínas Arqueales/química , Cristalografía por Rayos X/métodos , Guanosina/química , Nanoarchaeota/enzimología , ARN de Transferencia de Tirosina/química , Tirosina-ARNt Ligasa/química , Aminoacilación , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Modelos Moleculares , Elementos Estructurales de las Proteínas , ARN de Transferencia de Tirosina/genética , ARN de Transferencia de Tirosina/metabolismo , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismoRESUMEN
Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, ßLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant ßL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the ßHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.
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Bacillaceae/enzimología , Hidroliasas/metabolismo , Nitrilos/metabolismo , Piridinas/metabolismo , Proteínas Recombinantes/metabolismo , Sitios de Unión , Hidroliasas/química , Hidroliasas/aislamiento & purificación , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Nitrilos/química , Elementos Estructurales de las Proteínas , Piridinas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein-protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein-protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders.
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Fosfotransferasas (Aceptor de Grupo Alcohol) , Sitios de Unión , Dineínas Citoplasmáticas/metabolismo , Humanos , Mutación Missense , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Polimorfismo de Nucleótido Simple , Unión Proteica , Dominios Proteicos , Elementos Estructurales de las Proteínas , Relación Estructura-ActividadRESUMEN
Sea urchins are long-living marine invertebrates with a complex innate immune system, which includes expanded families of immune receptors. A central immune gene family in sea urchins encodes the Transformer (Trf) proteins. The Trf family has been studied mainly in the purple sea urchin Strongylocentrotus purpuratus. Here, we explore this protein family in the Mediterranean Sea urchin Paracentrotus lividus. The PlTrf genes and predicted proteins are highly diverse and show a typical Trf size range and structure. Coelomocytes and cell-free coelomic fluid from P. lividus contain different PlTrf protein repertoires with a shared subset, that bind specifically to E. coli. Using FACS, we identified five different P. lividus coelomocyte sub-populations with cell surface PlTrf protein expression. The relative abundance of the PlTrf-positive cells increases sharply following immune challenge with E. coli, but not following challenge with LPS or the sea urchin pathogen, Vibrio penaeicida. Phagocytosis of E. coli by P. lividus phagocytes is mediated through the cell-free coelomic fluid and is inhibited by blocking PlTrf activity with anti-SpTrf antibodies. Together, our results suggest a collaboration between cellular and humoral PlTrf-mediated effector arms in the P. lividus specific immune response to pathogens.
Asunto(s)
Inmunidad Celular , Inmunidad Humoral , Paracentrotus/inmunología , Fagocitosis , Proteínas Similares a la Proteína de Unión a TATA-Box/inmunología , Proteínas Similares a la Proteína de Unión a TATA-Box/metabolismo , Secuencia de Aminoácidos , Animales , Escherichia coli , Evolución Molecular , Paracentrotus/genética , Paracentrotus/microbiología , Fagocitos/inmunología , Fagocitos/metabolismo , Fagocitos/microbiología , Filogenia , Conformación Proteica , Elementos Estructurales de las Proteínas , Alineación de Secuencia , Proteínas Similares a la Proteína de Unión a TATA-Box/química , Proteínas Similares a la Proteína de Unión a TATA-Box/genética , VibrioRESUMEN
BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.
Asunto(s)
Anticuerpos Monoclonales/química , Cadenas Pesadas de Inmunoglobulina/química , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Anticuerpos de Dominio Único/química , Microglobulina beta-2/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Elementos Estructurales de las Proteínas , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Microglobulina beta-2/inmunologíaRESUMEN
BACKGROUND: Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of ESKD in individuals with type 1 diabetes at advanced kidney disease stage. METHODS: Gene-based exome array analyses of 15,449 genes in five large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time to ESKD, testing the top gene in a sixth cohort (n=2372/1115 events all cohorts) and replicating in two retrospective case-control studies (n=1072 cases, 752 controls). Deep resequencing of the top associated gene in five cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. RESULTS: Protein coding variants in the hydroxysteroid 17-ß dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (n=4196; P value=3.3 × 10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other CKD-associated renal pathologies. CONCLUSIONS: HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Nefropatías Diabéticas/genética , Fallo Renal Crónico/genética , Adulto , Animales , Estudios de Casos y Controles , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/metabolismo , Progresión de la Enfermedad , Exoma , Femenino , Expresión Génica , Variación Genética , Humanos , Fallo Renal Crónico/etiología , Fallo Renal Crónico/metabolismo , Túbulos Renales Proximales/enzimología , Masculino , Ratones , Persona de Mediana Edad , Elementos Estructurales de las Proteínas/genética , Daño por Reperfusión/complicaciones , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-ß-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.