RESUMEN
Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P2. The PI(4,5)P2-induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P2. CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/metabolismo , Lisina/metabolismo , Sitios de Unión , Lípidos , Unión ProteicaRESUMEN
Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.
Asunto(s)
Perfilación de la Expresión Génica , Enfermedades Renales , Riñón , Análisis de la Célula Individual , Transcriptoma , Humanos , Núcleo Celular/genética , Riñón/citología , Riñón/lesiones , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Transcriptoma/genética , Estudios de Casos y Controles , Imagenología TridimensionalRESUMEN
PIP3-dependent Rac exchanger 1 (P-Rex1) is activated downstream of G protein-coupled receptors to promote neutrophil migration and metastasis. The structure of more than half of the enzyme and its regulatory G protein binding site are unknown. Our 3.2 Å cryo-EM structure of the P-Rex1-Gßγ complex reveals that the carboxyl-terminal half of P-Rex1 adopts a complex fold most similar to those of Legionella phosphoinositide phosphatases. Although catalytically inert, the domain coalesces with a DEP domain and two PDZ domains to form an extensive docking site for Gßγ. Hydrogen-deuterium exchange mass spectrometry suggests that Gßγ binding induces allosteric changes in P-Rex1, but functional assays indicate that membrane localization is also required for full activation. Thus, a multidomain assembly is key to the regulation of P-Rex1 by Gßγ and the formation of a membrane-localized scaffold optimized for recruitment of other signaling proteins such as PKA and PTEN.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Membrana Celular/metabolismo , Movimiento Celular/fisiología , Microscopía por Crioelectrón/métodos , Humanos , Fosfohidrolasa PTEN/metabolismo , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Alineación de SecuenciaRESUMEN
Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, ß-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC50(insulin)â¯=â¯14â¯nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection.
Asunto(s)
Azoles/farmacología , Reposicionamiento de Medicamentos , Inhibidores Enzimáticos/farmacología , Insulisina/antagonistas & inhibidores , Compuestos de Organoselenio/farmacología , Azoles/química , Biocatálisis , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Insulisina/metabolismo , Isoindoles , Estructura Molecular , Compuestos de Organoselenio/química , Relación Estructura-ActividadRESUMEN
Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported â¼89â¯800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (â¼78â¯900 centroids), giving â¼100% coverage, and â¼10â¯900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σxÌ ). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.
Asunto(s)
Anticuerpos Monoclonales/química , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Fragmentos Fab de Inmunoglobulinas/análisisRESUMEN
Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.
Asunto(s)
Anticuerpos Neutralizantes/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Fragmentos Fab de Inmunoglobulinas/química , Virus de la Influenza A/química , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Especificidad de Anticuerpos , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Microscopía por Crioelectrón , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.
Asunto(s)
Marburgvirus/química , Modelos Químicos , Fosfatidilcolinas/química , Fosfatidilserinas/química , Multimerización de Proteína , Proteínas de la Matriz Viral/química , Medición de Intercambio de Deuterio , Marburgvirus/genética , Marburgvirus/metabolismo , Espectrometría de Masas , Estructura Cuaternaria de Proteína , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismoRESUMEN
Marburg virus (MARV) is a highly pathogenic filovirus that is classified in a genus distinct from that of Ebola virus (EBOV) (genera Marburgvirus and Ebolavirus, respectively). Both viruses produce a multifunctional protein termed VP35, which acts as a polymerase cofactor, a viral protein chaperone, and an antagonist of the innate immune response. VP35 contains a central oligomerization domain with a predicted coiled-coil motif. This domain has been shown to be essential for RNA polymerase function. Here we present crystal structures of the MARV VP35 oligomerization domain. These structures and accompanying biophysical characterization suggest that MARV VP35 is a trimer. In contrast, EBOV VP35 is likely a tetramer in solution. Differences in the oligomeric state of this protein may explain mechanistic differences in replication and immune evasion observed for MARV and EBOV. IMPORTANCE: Marburg virus can cause severe disease, with up to 90% human lethality. Its genome is concise, only producing seven proteins. One of the proteins, VP35, is essential for replication of the viral genome and for evasion of host immune responses. VP35 oligomerizes (self-assembles) in order to function, yet the structure by which it assembles has not been visualized. Here we present two crystal structures of this oligomerization domain. In both structures, three copies of VP35 twist about each other to form a coiled coil. This trimeric assembly is in contrast to tetrameric predictions for VP35 of Ebola virus and to known structures of homologous proteins in the measles, mumps, and Nipah viruses. Distinct oligomeric states of the Marburg and Ebola virus VP35 proteins may explain differences between them in polymerase function and immune evasion. These findings may provide a more accurate understanding of the mechanisms governing VP35's functions and inform the design of therapeutics.
Asunto(s)
Marburgvirus/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Estabilidad Proteica , Termodinámica , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The treatment of aqueous solutions of plasmid DNA with the protein avidin results in significant changes in physical, chemical, and biochemical properties. These effects include increased light scattering, formation of micron-sized particles containing both DNA and protein, and plasmid protection against thermal denaturation, radical attack, and nuclease digestion. All of these changes are consistent with condensation of the plasmid by avidin. Avidin can be displaced from the plasmid at higher ionic strengths. Avidin is not displaced from the plasmid by an excess of a tetra-arginine ligand, nor by the presence of biotin. Therefore, this system offers the opportunity to reversibly bind biotin-labeled species to a condensed DNA-protein complex. An example application is the use of biotinylated gold nanoparticles. This system offers the ability to examine in better detail the chemical mechanisms involved in important radiobiological effects. Examples include protein modulation of radiation damage to DNA, and radiosensitization by gold nanoparticles.
Asunto(s)
Avidina/química , Biotina/química , ADN/química , Oro/química , Nanopartículas del Metal/química , Protectores contra Radiación/química , Radioisótopos de Cesio , ADN/efectos de la radiación , Daño del ADN , PlásmidosRESUMEN
PURPOSE: Electron deficient guanine radical species are major intermediates produced in DNA by the direct effect of ionizing irradiation. There is evidence that they react with amine groups in closely bound ligands to form covalent crosslinks. Crosslink formation is very poorly characterized in terms of quantitative rate and yield data. We sought to address this issue by using oligo-arginine ligands to model the close association of DNA and its binding proteins in chromatin. MATERIALS AND METHODS: Guanine radicals were prepared in plasmid DNA by single electron oxidation. The product distribution derived from them was assayed by strand break formation after four different post-irradiation incubations. RESULTS: We compared the yields of DNA damage produced in the presence of four ligands in which neither, one, or both of the amino and carboxylate termini were blocked with amides. Free carboxylate groups were unreactive. Significantly higher yields of heat labile sites were observed when the amino terminus was unblocked. The rate of the reaction was characterized by diluting the unblocked amino group with its amide blocked derivative. CONCLUSION: These observations provide a means to develop quantitative estimates for the yields in which these labile sites are formed in chromatin by exposure to ionizing irradiation.
Asunto(s)
Aminas/metabolismo , ADN/metabolismo , Guanina/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Cromatina/metabolismo , ADN/química , ADN/genética , Roturas del ADN de Cadena Simple/efectos de la radiación , Transporte de Electrón/efectos de la radiación , Radicales Libres/metabolismo , Ligandos , Plásmidos/metabolismoRESUMEN
The DNA-binding proteins that are present in chromatin significantly affect the sensitivity of cells to ionizing radiation and to the radiation chemistry of DNA damage. The interaction between protein and DNA modifies the radiation chemistry of the latter. To model these processes, we have examined the effects of ionizing radiation on the minichromosome form of SV40 (which contains histone proteins arranged in nucleosomes) and also on plasmid DNA in the presence of lysozyme. Although high concentrations of lysozyme can bring about an extensive radioprotection by condensation of the plasmid, at lower levels it still produces significant radioprotective effects under conditions where this associative phase separation does not take place. The presence of histones or of lysozyme decreases the yield of modified guanines produced by ionizing radiation. Comparison with previous observations made with oligopeptides suggests that the mechanism responsible is electron donation to guanyl radicals in the DNA by tryptophan and tyrosine residues in the proteins. However, there was no evidence for DNA-protein crosslink formation.