RESUMO
Cotton wool plaques (CWPs) have been described as features of the neuropathologic phenotype of dominantly inherited Alzheimer disease (DIAD) caused by some missense and deletion mutations in the presenilin 1 (PSEN1) gene. CWPs are round, eosinophilic amyloid-ß (Aß) plaques that lack an amyloid core and are recognizable, but not fluorescent, in Thioflavin S (ThS) preparations. Amino-terminally truncated and post-translationally modified Aß peptide species are the main component of CWPs. Tau immunopositive neurites may be present in CWPs. In addition, neurofibrillary tangles coexist with CWPs. Herein, we report the structure of Aß and tau filaments isolated from brain tissue of individuals affected by DIAD caused by the PSEN1 V261I and A431E mutations, with the CWP neuropathologic phenotype. CWPs are predominantly composed of type I Aß filaments present in two novel arrangements, type Ic and type Id; additionally, CWPs contain type I and type Ib Aß filaments. Tau filaments have the AD fold, which has been previously reported in sporadic AD and DIAD. The formation of type Ic and type Id Aß filaments may be the basis for the phenotype of CWPs. Our data are relevant for the development of PET imaging methodologies to best detect CWPs in DIAD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Placa Amiloide , Presenilina-1 , Proteínas tau , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Humanos , Placa Amiloide/patologia , Placa Amiloide/metabolismo , Proteínas tau/metabolismo , Proteínas tau/genética , Peptídeos beta-Amiloides/metabolismo , Presenilina-1/genética , Encéfalo/patologia , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagem , Mutação , Feminino , MasculinoRESUMO
Adult individuals with Down syndrome (DS) develop Alzheimer disease (AD). Whether there is a difference between AD in DS and AD regarding the structure of amyloid-ß (Aß) and tau filaments is unknown. Here we report the structure of Aß and tau filaments from two DS brains. We found two Aß40 filaments (types IIIa and IIIb) that differ from those previously reported in sporadic AD and two types of Aß42 filaments (I and II) identical to those found in sporadic and familial AD. Tau filaments (paired helical filaments and straight filaments) were identical to those in AD, supporting the notion of a common mechanism through which amyloids trigger aggregation of tau. This knowledge is important for understanding AD in DS and assessing whether adults with DS could be included in AD clinical trials.
Assuntos
Peptídeos beta-Amiloides , Microscopia Crioeletrônica , Síndrome de Down , Proteínas tau , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Humanos , Proteínas tau/metabolismo , Proteínas tau/química , Proteínas tau/ultraestrutura , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/química , Adulto , Modelos MolecularesRESUMO
Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA-a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution.
Assuntos
Aminoácidos , Vírus , Humanos , Animais , Microscopia Crioeletrônica , Macaca mulatta , MutaçãoRESUMO
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable morbidity and mortality worldwide. Although authorized COVID-19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine-elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, a pseudovirus nanoparticle (PVNP) displaying the receptor binding domains (RBDs) of SARS-CoV-2 spike, named S-RBD, was generated and shown it as a promising COVID-19 vaccine candidate. The S-RBD PVNP was produced using both prokaryotic and eukaryotic systems. A 3D structural model of the S-RBD PVNPs was built based on the known structures of the S60 particle and RBDs, revealing an S60 particle-based icosahedral symmetry with multiple surface-displayed RBDs that retain authentic conformations and receptor-binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD-specific IgG and neutralizing antibodies in mice. The S-RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18-hACE2 mice from mortality and weight loss after a lethal SARS-CoV-2 challenge, supporting the S-RBD PVNPs as a potent COVID-19 vaccine candidate. By contrast, a PVNP displaying the N-terminal domain (NTD) of SARS-CoV-2 spike exhibited only 50% protective efficacy. Since the RBD antigens of our PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S-RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, these non-replicating PVNPs offer a flexible platform for a safe, effective COVID-19 vaccine with minimal manufacturing cost and time.
Assuntos
COVID-19 , Nanopartículas , Animais , Humanos , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Vacinas contra COVID-19/genética , Pandemias , Redução de PesoAssuntos
Doença de Alzheimer , Demência Frontotemporal , Tauopatias , Substância Branca , Humanos , Demência Frontotemporal/genética , Substância Branca/metabolismo , Tauopatias/complicações , Tauopatias/genética , Proteínas tau/genética , Proteínas tau/metabolismo , Encéfalo/metabolismo , Proteínas de Membrana , Proteínas do Tecido NervosoRESUMO
Background: The Microtubule-Associated Protein Tau (MAPT) is one of the proteins that are central to neurodegenerative diseases. The nature of intracellular tau aggregates is determined by the cell types whether neuronal or glial, the participating tau isoforms, and the structure of the amyloid filament. The transmembrane protein 106B (TMEM106B) has recently emerged as another significant player in neurodegeneration and aging. In the central nervous system, the composition of the gray and white matter differs considerably. The gray matter consists of nerve cell bodies, dendrites, unmyelinated axons, synaptic terminals, astrocytes, oligodendrocytes (satellite cells) and microglia. The white matter differs from the gray for the presence of axonal tracts as the only neuronal component and for the absence of nerve cell bodies, dendrites and synaptic terminals. Cryogenic electron microscopy (cryo-EM) studies have unveiled the structure of tau and TMEM106B, from the cerebral cortex, in several neurodegenerative diseases; however, whether tau and TMEM106B filaments from the gray and white matter share a common fold requires additional investigation. Methods: We isolated tau and TMEM106B from the cerebral cortex and white matter of the frontal lobes of two individuals affected by multiple system tauopathy with presenile dementia (MSTD), a disease caused by the MAPT intron 10 mutation +3. We used immunostaining, biochemical, genetics and cryo-EM methods to characterize tau and TMEM106B. Results: We determined that tau filaments in the gray and the white matter of MSTD individuals can induce tau aggregation and have identical AGD type 2 folds. TMEM106B amyloid filaments were also found in the gray and white matter of MSTD; the filament folds were identical in the two anatomical regions. Conclusions: Our findings show for the first time that in MSTD two types of amyloid filaments extracted from the gray matter have identical folds to those extracted from the white matter. Whether in this genetic disorder there is a relationship in the pathogenesis of the tau and TMEM106B filaments, remains to be determined. Furthermore, additional studies are needed for other proteins and other neurodegenerative diseases to establish whether filaments extracted from the gray and white matter would have identical folds.
RESUMO
Prion protein (PrP) aggregation and formation of PrP amyloid (APrP) are central events in the pathogenesis of prion diseases. In the dominantly inherited prion protein amyloidosis known as Gerstmann-Sträussler-Scheinker (GSS) disease, plaques made of PrP amyloid are present throughout the brain. The c.593t > c mutation in the prion protein gene (PRNP) results in a phenylalanine to serine amino acid substitution at PrP residue 198 (F198S) and causes the most severe amyloidosis among GSS variants. It has been shown that neurodegeneration in this disease is associated with the presence of extracellular APrP plaques and neuronal intracytoplasmic Tau inclusions, that have been shown to contain paired helical filaments identical to those found in Alzheimer disease. Using cryogenic electron microscopy (cryo-EM), we determined for the first time the structures of filaments of human APrP, isolated post-mortem from the brain of two symptomatic PRNP F198S mutation carriers. We report that in GSS (F198S) APrP filaments are composed of dimeric, trimeric and tetrameric left-handed protofilaments with their protomers sharing a common protein fold. The protomers in the cross-ß spines consist of 62 amino acids and span from glycine 80 to phenylalanine 141, adopting a previously unseen spiral fold with a thicker outer layer and a thinner inner layer. Each protomer comprises nine short ß-strands, with the ß1 and ß8 strands, as well as the ß4 and ß9 strands, forming a steric zipper. The data obtained by cryo-EM provide insights into the structural complexity of the PrP filament in a dominantly inherited human PrP amyloidosis. The novel findings highlight the urgency of extending our knowledge of the filaments' structures that may underlie distinct clinical and pathologic phenotypes of human neurodegenerative diseases.
Assuntos
Amiloidose , Doença de Gerstmann-Straussler-Scheinker , Príons , Amiloide/metabolismo , Amiloidose/metabolismo , Encéfalo/patologia , Microscopia Crioeletrônica , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Humanos , Fenilalanina/metabolismo , Placa Amiloide/patologia , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Príons/genética , Príons/metabolismo , Subunidades Proteicas/metabolismoRESUMO
In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann-Sträussler-Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments.
Assuntos
Doença de Alzheimer/metabolismo , Amiloidose/metabolismo , Emaranhados Neurofibrilares/patologia , Placa Amiloide/patologia , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Amiloidose/complicações , Encéfalo/patologia , Degeneração Corticobasal/metabolismo , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Humanos , Fenótipo , Placa Amiloide/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismoRESUMO
Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.
Assuntos
Anticorpos , Microscopia Crioeletrônica , Humanos , Ligantes , Fenômenos FísicosRESUMO
JSPR is a single particle cryo-EM image processing and 3D reconstruction software developed in the Jiang laboratory at Purdue University. It began as a few refinement scripts for symmetric and asymmetric reconstructions of icosahedral viruses, but has grown into a comprehensive suite of tools for building ab initio reconstructions, high resolution refinements of viruses, protein complexes of arbitrary symmetries including helical tubes/filaments, and image file handling utilities. In this review, we will present examples achieved using JSPR and demonstrate recently implemented features of JSPR such as multi-aberration "alignments" and automatic optimization of masking for the assessment of map resolution using "true" FSC.
Assuntos
Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Vírus/química , Bacteriófagos/química , Enterovirus/química , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Conformação Proteica , Software , Zika virus/químicaRESUMO
Encapsulin is a class of nanocompartments that is unique in bacteria and archaea to confine enzymatic activities and sequester toxic reaction products. Here we present a 2.87 Å resolution cryo-EM structure of Thermotoga maritima encapsulin with heterologous protein complex loaded. It is the first successful case of expressing encapsulin and heterologous cargo protein in the insect cell system. Although we failed to reconstruct the cargo protein complex structure due to the signal interference of the capsid shell, we were able to observe some unique features of the cargo-loaded encapsulin shell, for example, an extra density at the fivefold pore that has not been reported before. These results would lead to a more complete understanding of the encapsulin cargo assembly process of T. maritima.
Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Complexos Multiproteicos/ultraestrutura , Nanoestruturas/ultraestrutura , Thermotoga maritima/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nanoestruturas/química , Tamanho da Partícula , Porosidade , Multimerização Proteica , Estrutura Quaternária de Proteína , Thermotoga maritima/metabolismoRESUMO
Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87â¯Å resolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5â¯Å resolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM.
Assuntos
Apoferritinas/química , Microscopia Crioeletrônica/métodos , Coleta de Dados , Conformação ProteicaRESUMO
Phospholipase C (PLC) enzymes produce second messengers that increase the intracellular Ca2+ concentration and activate protein kinase C (PKC). These enzymes also share a highly conserved arrangement of core domains. However, the contributions of the individual domains to regulation are poorly understood, particularly in isoforms lacking high-resolution information, such as PLCϵ. Here, we used small-angle X-ray scattering (SAXS), EM, and functional assays to gain insights into the molecular architecture of PLCϵ, revealing that its PH domain is conformationally dynamic and essential for activity. We further demonstrate that the PH domain of PLCß exhibits similar dynamics in solution that are substantially different from its conformation observed in multiple previously reported crystal structures. We propose that this conformational heterogeneity contributes to subfamily-specific differences in activity and regulation by extracellular signals.
Assuntos
Simulação de Dinâmica Molecular , Domínios de Homologia à Plecstrina , Fosfolipases Tipo C/química , Animais , Humanos , Mutação , Ratos , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Homotypic interactions of viral capsid proteins are common, driving viral capsid self-formation. By taking advantage of such interactions of the norovirus shell (S) domain that naturally builds the interior shells of norovirus capsids, we have developed a technology to produce 60-valent, icosahedral S60 nanoparticles through the E. coli system. This has been achieved by several modifications to the S domain, including an R69A mutation to destruct an exposed proteinase cleavage site and triple cysteine mutations (V57C/Q58C/S136C) to establish inter-S domain disulfide bonds for enhanced inter-S domain interactions. The polyvalent S60 nanoparticle with 60 exposed S domain C-termini offers an ideal platform for antigen presentation, leading to enhanced immunogenicity to the surface-displayed antigens for vaccine development. This was proven by constructing a chimeric S60 nanoparticle displaying 60 rotavirus (RV) VP8* proteins, the major RV-neutralizing antigen. These S60-VP8* particles are easily produced and elicited high IgG response in mice toward the displayed VP8* antigens. The mouse antisera after immunization with the S60-VP8* particles exhibited high blockades against RV VP8* binding to its glycan ligands and high neutralizing activities against RV infection in culture cells. The three-dimensional structures of the S60 and S60-VP8* particles were studied. Furthermore, the S60 nanoparticle can display other antigens, supporting the notion that the S60 nanoparticle is a multifunctional vaccine platform. Finally, the intermolecular disulfide bond approach may be used to stabilize other viral-like particles to display foreign antigens for vaccine development.
Assuntos
Bioengenharia , Nanopartículas/química , Rotavirus/imunologia , Vacinas Virais/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Rotavirus/isolamento & purificação , Vacinas Virais/químicaRESUMO
First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary σ70 factor forms the RNAP holoenzyme to express housekeeping genes. The σ70 contains a large insertion between the conserved regions 1.2 and 2.1, the σ non-conserved region (σNCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP σ70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 Å resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between the σNCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography because of restraint of crystal packing effects. Disruption of the σNCR and DNA interaction by the amino acid substitutions (R157A/R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the σNCR and DNA interaction is conserved in proteobacteria, and RNAP in other bacteria replaces its role with a transcription factor.
Assuntos
Microscopia Crioeletrônica/métodos , DNA Bacteriano/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Regiões Promotoras Genéticas , Fator sigma/química , DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Genes Essenciais , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Fator sigma/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160-166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104-135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.
Assuntos
Proteínas de Fímbrias/química , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
EpsH is a minor pseudopilin protein of the Vibrio cholerae type II secretion system. A truncated form of EpsH with a C-terminal noncleavable His tag was constructed and expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 1.71 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.39, b = 71.11, c = 84.64 A. There were two protein molecules in the asymmetric unit, which gave a Matthews coefficient V(M) of 2.1 A(3) Da(-1), corresponding to 41.5% solvent content.