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Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.
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Nanopartículas , Vacunas Virales , Humanos , Nanopartículas/química , Animales , Vacunas Virales/inmunología , Virosis/prevención & control , Virosis/inmunología , Virus/inmunología , Virus/genética , Antígenos Virales/inmunología , Antígenos Virales/genética , Vacunas de Subunidad/inmunologíaRESUMEN
Aberrant aggregation of α-Synuclein is the pathological hallmark of a set of neurodegenerative diseases termed synucleinopathies. Recent advances in cryo-electron microscopy have led to the structural determination of the first synucleinopathy-derived α-Synuclein fibrils, which contain a non-proteinaceous, "mystery density" at the core of the protofilaments, hypothesized to be highly negatively charged. Guided by previous studies that demonstrated that polyphosphate (polyP), a universally conserved polyanion, significantly accelerates α-Synuclein fibril formation, we conducted blind docking and molecular dynamics simulation experiments to model the polyP binding site in α-Synuclein fibrils. Here we demonstrate that our models uniformly place polyP into the lysine-rich pocket, which coordinates the mystery density in patient-derived fibrils. Subsequent in vitro studies and experiments in cells revealed that substitution of the two critical lysine residues K43 and K45 leads to a loss of all previously reported effects of polyP binding on α-Synuclein, including stimulation of fibril formation, change in filament conformation and stability as well as alleviation of cytotoxicity. In summary, our study demonstrates that polyP fits the unknown electron density present in in vivo α-Synuclein fibrils and suggests that polyP exerts its functions by neutralizing charge repulsion between neighboring lysine residues.
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The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.
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Proteínas de Unión al ADN , G-Cuádruplex , Agregado de Proteínas , Humanos , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Transición de Fase , Unión Proteica , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Mensajero/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismoRESUMEN
Human small EDRK-rich factor protein SERF2 is a cellular driver of protein amyloid formation, a process that has been linked to neurodegenerative diseases including Alzheimer's and Parkinson's disease. SERF2 is a 59 amino acid protein, highly charged, and well conserved whose structure and physiological function is unclear. SERF family proteins including human SERF2 have shown a tendency to form fuzzy complexes with misfolded proteins such as α-Synuclein which has been linked to Parkinson's disease. SERF family proteins have been recently identified to bind nucleic acids, but the binding mechanism(s) remain enigmatic. Here, using multidimensional solution NMR, we report the 1H, 15N, and 13C chemical shift assignments (~ 86% of backbone resonance assignments) for human SERF2. TALOS-N predicted secondary structure of SERF2 showed three very short helices (3-4 residues long) in the N-terminal region of the protein and a long helix in the C-terminal region spanning residues 37-46 which is consistent with the helical content indicated by circular dichroism spectroscopy. Paramagnetic relaxation enhancement NMR analysis revealed that a short C-terminal region E53-K55 is in the proximity of the N-terminus. Having the backbone assignment of SERF2 allowed us to probe its interaction with α-Synuclein and to identify the residues in SERF2 binding interfaces that likely promote α-Synuclein aggregation.
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Resonancia Magnética Nuclear Biomolecular , Humanos , Secuencia de Aminoácidos , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706's ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.
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The catalytic activity of cytochrome P450 2B4 (CYP2B4) is moderated by its cognate redox partner cytochrome b5 (Cyt-b5). The endoplasmic reticulum (ER) membrane and intermolecular transmembrane (TM) interaction between CYP2B4 and Cyt-b5 regulate the substrate catalysis and the reaction rate. This emphasizes the significance of elucidating the molecular basis of CYP2B4 and Cyt-b5 complexation in a membrane environment to better understand the enzymatic activity of CYP2B4. Our previous solid-state NMR studies revealed the membrane topology of the transmembrane domains of these proteins in the free and complex forms. Here, we show the cross-angle complex formation by the single-pass TM domains of CYP2B4 and Cyt-b5, which is mainly driven by several salt-bridges (E2-R128, R21-D104 and K25-D104), using a multi-microsecond molecular dynamic simulation. Additionally, the leucine-zipper residues (L8, L12, L15, L18 and L19 from CYP2B4) and π-stacking between H23 and F20 residues of CYP2B4 and W110 of Cyt-b5 are identified to stabilize the TM-TM complex in the ER membrane. The simulated tilts of the helices in the free and in the complex are in excellent agreement with solid-state NMR results. The TM-TM packing influences a higher order structural stability when compared to the complex formed by the truncated soluble domains of these two proteins. MM/PBSA based binding free energy estimates nearly 100-fold higher binding affinity (ΔG = -2810.68 ± 696.44 kJ/mol) between the soluble domains of the full-length CYP2B4 and Cyt-b5 when embedded in lipid membrane as compared to the TM-domain-truncated soluble domains (ΔG = -27.406 ± 10.32 kJ/mol). The high-resolution full-length CYP2B4-Cyt-b5 complex structure and its dynamics in a native ER membrane environment reported here could aid in the development of approaches to effectively modulate the drug-metabolism activity of CYP2B4.
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Hidrocarburo de Aril Hidroxilasas , Citocromos b5 , Citocromos b5/química , Citocromos b5/metabolismo , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Familia 2 del Citocromo P450/metabolismo , Oxidación-ReducciónRESUMEN
The severe consequences of the Zika virus (ZIKV) infections resulting in congenital Zika syndrome in infants and the autoimmune Guillain-Barre syndrome in adults warrant the development of safe and efficacious vaccines and therapeutics. Currently, there are no approved treatment options for ZIKV infection. Herein, we describe the development of a bacterial ferritin-based nanoparticle vaccine candidate for ZIKV. The viral envelope (E) protein domain III (DIII) was fused in-frame at the amino-terminus of ferritin. The resulting nanoparticle displaying the DIII was examined for its ability to induce immune responses and protect vaccinated animals upon lethal virus challenge. Our results show that immunization of mice with a single dose of the nanoparticle vaccine candidate (zDIII-F) resulted in the robust induction of neutralizing antibody responses that protected the animals from the lethal ZIKV challenge. The antibodies neutralized infectivity of other ZIKV lineages indicating that the zDIII-F can confer heterologous protection. The vaccine candidate also induced a significantly higher frequency of interferon (IFN)-γ positive CD4 T cells and CD8 T cells suggesting that both humoral and cell-mediated immune responses were induced by the vaccine candidate. Although our studies showed that a soluble DIII vaccine candidate could also induce humoral and cell-mediated immunity and protect from lethal ZIKV challenge, the immune responses and protection conferred by the nanoparticle vaccine candidate were superior. Further, passive transfer of neutralizing antibodies from the vaccinated animals to naïve animals protected against lethal ZIKV challenge. Since previous studies have shown that antibodies directed at the DIII region of the E protein do not to induce antibody-dependent enhancement (ADE) of ZIKV or other related flavivirus infections, our studies support the use of the zDIII-F nanoparticle vaccine candidate for safe and enhanced immunological responses against ZIKV.
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Viral infections alter host cell metabolism and homeostasis; however, the mechanisms that regulate these processes have only begun to be elucidated. We report here that Zika virus (ZIKV) infection activates the antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2), which precedes oxidative stress. Downregulation of Nrf2 or inhibition of glutathione (GSH) synthesis resulted in significantly increased viral replication. Interestingly, 6-amino-nicotinamide (6-AN), a nicotinamide analog commonly used as an inhibitor of the pentose phosphate pathway (PPP), decreased viral replication by over 1,000-fold. This inhibition was neither recapitulated by the knockdown of PPP enzymes, glucose 6-phosphate dehydrogenase (G6PD), or 6-phosphogluconate dehydrogenase (6PGD), nor prevented by supplementation with ribose 5-phosphate. Instead, our metabolomics and metabolic phenotype studies support a mechanism in which 6-AN depletes cells of NAD(H) and impairs NAD(H)-dependent glycolytic steps resulting in inhibition of viral replication. The inhibitory effect of 6-AN was rescued with precursors of the salvage pathway but not with those of other NAD+ biosynthesis pathways. Inhibition of glycolysis reduced viral protein levels, which were recovered transiently. This transient recovery in viral protein synthesis was prevented when oxidative metabolism was inhibited by blockage of the mitochondrial pyruvate carrier, fatty acid oxidation, or glutaminolysis, demonstrating a compensatory role of mitochondrial metabolism in ZIKV replication. These results establish an antagonistic role for the host cell Nrf2/GSH/NADPH-dependent antioxidant response against ZIKV and demonstrate the dependency of ZIKV replication on NAD(H). Importantly, our work suggests the potential use of NAD(H) antimetabolite therapy against the viral infection. IMPORTANCE Zika virus (ZIKV) is a major public health concern of international proportions. While the incidence of ZIKV infections has declined substantially in recent years, the potential for the reemergence or reintroduction remains high. Although viral infection alters host cell metabolism and homeostasis to promote its replication, deciphering the mechanism(s) involved in these processes is important for identifying therapeutic targets. The present work reveals the complexities of host cell redox regulation and metabolic dependency of ZIKV replication. An antagonistic effect of the Nrf2/GSH/NADP(H)-dependent antioxidant response against ZIKV infection and an essential role of NAD(H) metabolism and glycolysis for viral replication are established for the first time. These findings highlight the potential use of NAD(H) antimetabolites to counter ZIKV infection and pathogenesis.
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Interacciones Microbiota-Huesped , Factor 2 Relacionado con NF-E2 , Replicación Viral , Infección por el Virus Zika , Virus Zika , Humanos , Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , NAD/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Virus Zika/fisiología , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virología , Oxidorreductasas/genética , Técnicas de Silenciamiento del Gen , Células Cultivadas , Interacciones Microbiota-Huesped/fisiologíaRESUMEN
Amyloid formation is a misfolding process that has been linked to age-related diseases, including Alzheimer's and Huntington's. Understanding how cellular factors affect this process in vivo is vital in realizing the dream of controlling this insidious process that robs so many people of their humanity. SERF (small EDRK-rich factor) was initially isolated as a factor that accelerated polyglutamine amyloid formation in a C. elegans model. SERF knockouts inhibit amyloid formation of a number of proteins that include huntingtin, α-synuclein and ß-amyloid which are associated with Huntington's, Parkinson's and Alzheimer's disease, respectively, and purified SERF protein speeds their amyloid formation in vitro. SERF proteins are highly conserved, highly charged and conformationally dynamic proteins that form a fuzzy complex with amyloid precursors. They appear to act by specifically accelerating the primary step of amyloid nucleation. Brain-specific SERF knockout mice, though viable, appear to be more prone to deposition of amyloids, and show modified fibril morphology. Whole-body knockouts are perinatally lethal due to an apparently unrelated developmental issue. Recently, it was found that SERF binds RNA and is localized to nucleic acid-rich membraneless compartments. SERF-related sequences are commonly found fused to zinc finger sequences. These results point towards a nucleic acid-binding function. How this function relates to their ability to accelerate amyloid formation is currently obscure. In this review, we discuss the possible biological functions of SERF family proteins in the context of their structural fuzziness, modulation of amyloid pathway, nucleic acid binding and their fusion to folded proteins.
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Enfermedad de Alzheimer , Ácidos Nucleicos , Ratones , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Amiloide/química , Péptidos beta-Amiloides/metabolismoRESUMEN
The fabrication of electrochemical sensing platforms for cancer monitoring by quantifying circulating tumor cells (CTCs) in blood holds promise for providing a low-cost, rapid, feasible, and safe approach for cancer diagnosis. Here, we isolate cancer cells using CoFe2O4 nanoparticles functionalized with folic acid and chitosan as an inexpensive magnetic nanoprobe. This electrochemical cytosensing platform was realized using polyaniline-folic acid nanohybrids with a three-dimensional hierarchical structure that presents abundant affinity sites toward overexpressed folate bioreceptors on cancer cells, in addition to retaining satisfied conductivity. Furthermore, 3D modeling and simulation of the polyaniline-folic acid structures were conducted to investigate the stable complex between aniline and folate, and the interaction between the polyaniline-folate complex and folate receptor alpha1, a bioreceptor on MCF-7 was revealed for the first time. The limit of detection was calculated to be 4 cells mL-1 with a linear range from 50 to 106 cells mL-1.
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Técnicas Biosensibles , Nanopartículas , Nanoestructuras , Ácido Fólico , Nanoestructuras/química , Nanopartículas/química , Compuestos de Anilina/química , Técnicas Biosensibles/métodos , Técnicas ElectroquímicasRESUMEN
A recently proposed lipid-chaperone hypothesis suggests that free lipid molecules, not bound to membranes, affect the aggregation of amyloidogenic peptides such as amyloid-ß (Aß) peptides, whose aggregates are the hallmarks of Alzheimer's disease. Here, we combine experiments with all-atom molecular dynamics simulations in explicit solvent to explore the effects of neuronal ganglioside GM1, abundant in mammalian brains, on the aggregation of two principal isoforms of Aß, Aß40 and Aß42. Our simulations show that free GM1 forms stable, highly water-soluble complexes with both isoforms, and nuclear magnetic resonance experiments support the formation of well-ordered, structurally compact GM1+Aß complexes. By simulation, we also show that Aß40 monomers display a preference for binding to GM1-containing hetero-oligomers over GM1-lacking homo-oligomers, while Aß42 monomers have the opposite preference. These observations explain why GM1 dose-dependently inhibits Aß40 aggregation but has no effect on Aß42 aggregation, as assessed by thioflavin T fluorescence.
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Enfermedad de Alzheimer , Gangliósido G(M1) , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Animales , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Gangliósidos/metabolismo , Mamíferos/metabolismo , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Solventes , AguaRESUMEN
Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in ß-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two ß-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.
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Polipéptido Amiloide de los Islotes Pancreáticos/química , Maleatos/química , Conformación Molecular , Estireno/química , Amiloide/química , Animales , Simulación por Computador , Diabetes Mellitus Tipo 2 , Fluorescencia , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Agregado de Proteínas , Espectroscopía Infrarroja por Transformada de Fourier , Estirenos/química , Pez CebraRESUMEN
Nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) recognizes the muramyl dipeptide and activates the NF-κB signaling cascade following its interaction with receptor-interacting protein 2 (RIP2) via caspase recruitment domains (CARDs). The NOD2-RIP2 interaction is not understood well due to inadequate structural information. Using comparative modeling and multimicrosecond timescale molecular dynamics simulations, we have demonstrated the association of NOD2-CARDs (CARDa-CARDb) and their interaction with RIP2CARD. Our results suggest that a negatively charged interface of NOD2CARDa and positively charged type-Ia interface of NOD2CARDb are crucial for CARDa-CARDb association and the type-Ia interface of NOD2CARDa and type-Ib interface of RIP2CARD predicted to be involved in 1:1 CARD-CARD interaction. Moreover, the direct interaction of NOD2CARDb with RIP2CARD signifies the importance of both CARDs of NOD2 in RIP2-mediated CARD-CARD interaction. Altogether, the structural results could help in understanding the underlying molecular details of the NOD2-RIP2 association in higher and lower eukaryotes.
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Simulación de Dinámica Molecular , FN-kappa B , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic peptide found in pancreatic islets of type-2 diabetes (T2D) patients. Under certain conditions, hIAPP is able to form amyloid fibrils that play a role in the progression of T2D. hIAPP is synthesized in the ß-cell of the pancreas and stored in the secretory granules before being released into the extracellular compartment. It has been suggested that natural stabilizing agents, such as insulin or zinc present in the secretory granules with hIAPP could prevent hIAPP fibril formation. The difference in the amino acid sequences of IAPP among species strongly correlates with amyloidogenicity and toxicity. The residue histidine at position 18 is known to be important in modulating the fibril formation, membrane leakage and toxicity. In this study, we have synthesized four analogues of hIAPP (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and characterized their aggregation with either insulin or zinc in order to determine the effect of the residue-18 on the insulin-IAPP and zinc-IAPP interactions using a variety of biophysical experiments including thioflavin-T fluorescence, transmission electron microscopy imaging, circular dichroism, and NMR spectroscopy. We show that insulin reduced hIAPP fibril formation both in solution and in the presence of membrane and hIAPP-membrane damage and that the interactions are somewhat mediated by the residue-18. In addition, our results reveal that zinc affects the process of hIAPP fibril formation in solution but not in the presence of membrane. Our results indicate that the nature of the residue-18 is important for zinc binding. Based on this observation, we hypothesize that zinc binds to the residues in the N-terminal region of hIAPP, which is not accessible in the presence of membrane due to its strong interaction with lipids.
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Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Agregado de Proteínas/fisiología , Liposomas Unilamelares/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Microscopía Electrónica de Transmisión , Unión Proteica , Espectrometría de Fluorescencia , Liposomas Unilamelares/químicaRESUMEN
It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-ß (Aß). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aß degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aß(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aß(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aß fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aß(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aß(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aß degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.
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Enfermedad de Alzheimer/metabolismo , Insulisina/metabolismo , Proteínas Mutantes/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Biocatálisis , Cromatografía Líquida de Alta Presión , Humanos , Insulisina/química , Insulisina/genética , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Unión Proteica , Proteolisis , Especificidad por Sustrato , Zinc/química , Zinc/metabolismoRESUMEN
Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control.
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Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro, in vivo, and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aß, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.
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Amiloide/química , Amiloide/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Polipéptido Amiloide de los Islotes Pancreáticos/química , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Modelos Moleculares , Enfermedades Neurodegenerativas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas , Deficiencias en la Proteostasis/metabolismo , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-ß, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-ß, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-ß, and α-synuclein) and nonamyloidogenic (rat IAPP, ß-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Amiloide , Proteínas Amiloidogénicas , Animales , Polipéptido Amiloide de los Islotes Pancreáticos , Lípidos , Ratas , alfa-SinucleínaRESUMEN
Zika virus (ZIKV), a mosquito-transmitted flavivirus, is linked to microcephaly and other neurological defects in neonates and Guillain-Barré syndrome in adults. The molecular mechanisms regulating ZIKV infection and pathogenic outcomes are incompletely understood. Signaling by the mechanistic (mammalian) target of rapamycin (mTOR) kinase is important for cell survival and proliferation, and viruses are known to hijack this pathway for their replication. Here, we show that in human neuronal precursors and glial cells in culture, ZIKV infection activates both mTOR complex 1 (mTORC1) and mTORC2. Inhibition of mTOR kinase by Torin1 or rapamycin results in reduction in ZIKV protein expression and progeny production. Depletion of Raptor, the defining subunit of mTORC1, by small interfering RNA (siRNA) negatively affects ZIKV protein expression and viral replication. Although depletion of Rictor, the unique subunit of mTORC2, or the mTOR kinase itself also inhibits the viral processes, the extent of inhibition is less pronounced. Autophagy is transiently induced early by ZIKV infection, and impairment of autophagosome elongation by the class III phosphatidylinositol 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA) enhances viral protein accumulation and progeny production. mTOR phosphorylates and inactivates ULK1 (S757) at later stages of ZIKV infection, suggesting a link between autophagy inhibition and mTOR activation by ZIKV. Accordingly, inhibition of ULK1 (by MRT68921) or autophagy (by 3-MA) reversed the effects of mTOR inhibition, leading to increased levels of ZIKV protein expression and progeny production. Our results demonstrate that ZIKV replication requires the activation of both mTORC1 and mTORC2, which negatively regulates autophagy to facilitate ZIKV replication.IMPORTANCE The re-emergence of Zika virus (ZIKV) and its association with neurological complications necessitates studies on the molecular mechanisms that regulate ZIKV pathogenesis. The mTOR signaling cascade is tightly regulated and central to normal neuronal development and survival. Disruption of mTOR signaling can result in neurological abnormalities. In the studies reported here, we demonstrate for the first time that ZIKV infection results in activation of both mTORC1 and mTORC2 to promote virus replication. Although autophagy is activated early in infection to counter virus replication, it is subsequently suppressed by mTOR. These results reveal critical roles of mTOR signaling and autophagy in ZIKV infection and point to a possible mechanism underlying ZIKV-induced pathogenesis. Elucidating the role of mTOR signaling in ZIKV infection will provide insights into the mechanisms of ZIKV-induced neurological complications and potential targets for therapeutic approaches.
Asunto(s)
Autofagia/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/efectos de los fármacos , Línea Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Virales , Virus Zika/fisiología , Infección por el Virus Zika/virologíaRESUMEN
Natural-abundance 17O NMR experiments are used to investigate the hydrated water in magnetically aligned synthetic polymer based lipid-nanodiscs. Residual quadrupole couplings (RQCs) measured from the observed five 17O (central and satellite) transitions, and molecular dynamics simulations, are used to probe the ordering of water molecules across the lipid bilayer.