RESUMO
Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.
Assuntos
Catarata , Mercúrio , gama-Cristalinas , Humanos , gama-Cristalinas/química , gama-Cristalinas/genética , gama-Cristalinas/metabolismo , Catarata/genética , Catarata/metabolismo , Mutação , ÍonsRESUMO
The fabrication of efficient organic electrochemical transistors (OECTs)-based biosensors requires the design of biocompatible interfaces for the immobilization of biorecognition elements, as well as the development of robust channel materials to enable the transduction of the biochemical event into a reliable electrical signal. In this work, PEDOT-polyamine blends are shown as versatile organic films that can act as both highly conducting channels of the transistors and non-denaturing platforms for the construction of the biomolecular architectures that operate as sensing surfaces. To achieve this goal, we synthesized and characterized films of PEDOT and polyallylamine hydrochloride (PAH) and employed them as conducting channels in the construction of OECTs. Next, we studied the response of the obtained devices to protein adsorption, using glucose oxidase (GOx) as a model system, through two different strategies: The direct electrostatic adsorption of GOx on the PEDOT-PAH film and the specific recognition of the protein by a lectin attached to the surface. Firstly, we used surface plasmon resonance to monitor the adsorption of the proteins and the stability of the assemblies on PEDOT-PAH films. Then, we monitored the same processes with the OECT showing the capability of the device to perform the detection of the protein binding process in real time. In addition, the sensing mechanisms enabling the monitoring of the adsorption process with the OECTs for the two strategies are discussed.
Assuntos
Técnicas Biossensoriais , Polímeros , Ligação Proteica , Polímeros/química , Glucose Oxidase/química , PoliaminasRESUMO
SARS-CoV-2 infection depend on the binding of the viral Spike glycoprotein (S) to the human receptor Angiotensin Converting Enzyme 2 (ACE2) to induce virus-cell membrane fusion. S protein evolved diverse amino acid changes that are possibly linked to more efficient binding to human ACE2, which might explain part of the increase in frequency of SARS-CoV-2 Variants Of Concern (VOCs). In this work, we investigated the role of ACE2 protein variations that are naturally found in human populations and its binding affinity with S protein from SARS-CoV-2 representative genotypes, based on a series of in silico approaches involving molecular modelling, docking and molecular dynamics simulations. Our results indicate that SARS-CoV-2 VOCs bind more efficiently to the human receptor ACE2 than the ancestral Wuhan genotype. Additionally, variations in the ACE2 protein can affect SARS-CoV-2 binding and protein-protein stability, mostly making the interaction weaker and unstable in some cases. We show that some VOCs, such as B.1.1.7 and P.1 are much less sensitive to ACE2 variants, while others like B.1.351 appear to be specifically optimized to bind to the widespread wild-type ACE2 protein.Communicated by Ramaswamy H. Sarma.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Enzima de Conversão de Angiotensina 2/química , Sítios de Ligação , Simulação de Dinâmica Molecular , Ligação Proteica , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Histatin-1 is a salivary peptide with antimicrobial and wound healing promoting activities, which was previously shown to stimulate angiogenesis in vitro and in vivo via inducing endothelial cell migration. The mechanisms underlying the proangiogenic effects of Histatin-1 remain poorly understood and specifically, the endothelial receptor for this peptide, is unknown. Based on the similarities between Histatin-1-dependent responses and those induced by the prototypical angiogenic receptor, vascular endothelial growth factor receptor 2 (VEGFR2), we hypothesized that VEGFR2 is the Histatin-1 receptor in endothelial cells. First, we observed that VEGFR2 is necessary for Histatin-1-induced endothelial cell migration, as shown by both pharmacological inhibition studies and siRNA-mediated ablation of VEGFR2. Moreover, Histatin-1 co-immunoprecipitated and co-localized with VEGFR2, associating spatial proximity between these proteins with receptor activation. Indeed, pulldown assays with pure, tagged and non-tagged proteins showed that Histatin-1 and VEGFR2 directly interact in vitro. Optical tweezers experiments permitted estimating kinetic parameters and rupture forces, indicating that the Histatin-1-VEGFR2 interaction is transient, but specific and direct. Sequence alignment and molecular modeling identified residues Phe26, Tyr30 and Tyr34 within the C-terminal domain of Histatin-1 as relevant for VEGFR2 binding and activation. This was corroborated by mutation and molecular dynamics analyses, as well as in direct binding assays. Importantly, these residues were required for Histatin-1 to induce endothelial cell migration and angiogenesis in vitro. Taken together, our findings reveal that VEGFR2 is the endothelial cell receptor of Histatin-1 and provide insights to the mechanism by which this peptide promotes endothelial cell migration and angiogenesis.
Assuntos
Células Endoteliais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Proteínas de Transporte/metabolismo , Movimento Celular , Células Endoteliais/metabolismo , Histatinas/metabolismo , Histatinas/farmacologia , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hemopexin (Hx) is a plasma glycoprotein that scavenges heme (Fe(III) protoporphyrin IX). Hx has important implications in hemolytic disorders and hemorrhagic conditions because releasing hemoglobin increases the labile heme, which is potentially toxic, thus producing oxidative stress. Therefore, Hx has been considered for therapeutic use and diagnostics. In this work, we analyzed and mapped the interaction sequences of Hx with hemin and hemoglobin. The spot-synthesis technique was used to map human hemopexin (P02790) binding to hemin and human hemoglobin. A library of 15 amino acid peptides with a 10-amino acid overlap was designed to represent the entire coding region (aa 1-462) of hemopexin and synthesized onto cellulose membranes. An in silico approach was taken to analyze the amino acid frequency in the identified interaction regions, and molecular docking was applied to assess the protein-protein interaction. Seven linear peptide sequences in Hx were identified to bind hemin (H1-H7), and five were described for Hb (Hb1-Hb5) interaction, with just two sequences shared between hemin and Hb. The amino acid composition of the identified sequences demonstrated that histidine residues are relevant for heme binding. H105, H293, H373, H400, H429, and H462 were distributed in the H1-H7 peptide sequences, but other residues may also play an important role. Molecular docking analysis demonstrated Hx's association with the ß-chain of Hb, with several hotspot amino acids that coordinated the interaction. This study provides new insights into Hx-hemin binding motifs and protein-protein interactions with Hb. The identified binding sequences and specific peptides can be used for therapeutic purposes and diagnostics as hemopexin is under investigation to treat different diseases and there is an urgent need for diagnostics using labile heme when monitoring hemolysis.
Assuntos
Hemina , Hemopexina , Compostos Férricos , Heme/metabolismo , Hemina/metabolismo , Hemoglobinas/metabolismo , Hemólise , Hemopexina/metabolismo , Histidina , Humanos , Simulação de Acoplamento MolecularRESUMO
Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular accumulation of amyloid-ß peptide (Aß) in the brain interstitium. Human serum albumin (HSA) highly binds to Aß in blood plasma and is thought to inhibit plaque formation in peripheral tissue. Thus, the evaluation of albumin binding to Aß is an important key to understand the dynamics of these molecules in the biological system of patients with AD. In this work, a fiber-in-tube solid-phase microextraction (fiber-in-tube SPME) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to estimate Aß fraction binding to HSA in cerebrospinal fluid (CSF) and plasma samples. Crosslinked zwitterionic polymeric ionic liquid (zwitterionic PIL)-coated nitinol wires were developed and packed into a polyether ether ketone (PEEK) capillary for a fiber-in-tube SPME and UHPLC-MS/MS method. Zwitterionic PIL sorbent was synthetized from 1-vinyl-3-(butanesulfonate)imidazolium ([VIm+C4SO3-]) and 1,12-di(3-vinylimidazolium)dodecane dibromide ([(VIm)2C12]2[Br]) monomers by in-situ thermally-initiated polymerization. Morphological characterization by scanning electron microscopy (SEM) and atomic force microscopy (AFM) revealed a decrease in the surface roughness of the nitinol wires from â¼17 nm to 1 nm after the in-situ polymerization. The zwitterionic PIL sorbent selectively preconcentrates Aß through a two-pronged interaction mechanism. The fiber-in-tube SPME and UHPLC-MS/MS method presented lower limits of quantification (LLOQ) of 0.4 ng mL-1 for Aß38 and 0.3 ng mL-1 for Aß40 and Aß42, a linear range from LLOQ values to 15 ng mL-1 with coefficients of determination higher than 0.99, precision with coefficient of variation (CV) values ranging from 2.1 to 7.3% and accuracy with relative standard deviation (RSD) values from -0.3 to 7.4. This method was successfully applied to evaluate the binding of HSA to Aß in cerebrospinal fluid (CSF) and plasma samples.
Assuntos
Peptídeos beta-Amiloides , Líquidos Iônicos , Ligas , Proteínas de Transporte , Cromatografia Líquida de Alta Pressão , Humanos , Microextração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.
Assuntos
COVID-19/etiologia , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , COVID-19/sangue , Hemina/metabolismo , Hemoglobinas/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteômica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Ligação Viral , Replicação ViralRESUMO
Fluorescent probes are used in drug nanocarrier pre-clinical studies or as active compounds in theranostics and photodynamic therapy. In the biological medium, nanoparticles interact with proteins, which can result in the off-target release of their cargo. The present study used asymmetric flow field-flow fractionation with online multi-angle laser light scattering and fluorescence detection (AF4-MALLS-FLD) to study the release, transfer, and partition of fluorescent dyes from polymeric nanoparticles (NP). NP formulations containing the dyes Rose Bengal, Rhodamine B, DiI, 3-(α-azidoacetyl)coumarin and its polymer conjugate, Nile Red, and IR780 and its polymer conjugate were prepared. NP suspensions were incubated in a medium with serum proteins and then analyzed by AF4. AF4 allowed efficient separation of proteins (< 10 nm) from fluorescently labeled NP (range of 54 - 180 nm in diameters). The AF4 analyses showed that some dyes, such as Rose Bengal, IR780, and Coumarin were transferred to a high extent (68-77%) from NP to proteins. By contrast, for DiI and dye-polymer conjugates, transfer occured to a lower extent. The studies of dye release kinetics showed that the transfer of IR780 from NP to proteins occurs at a high extent (~50%) and rate, while Nile Red was slowly released from the NP over time with reduced association with proteins (~20%). This experiment assesses the stability of fluorescence labeling of nanocarriers and probes the transfer of fluorescent dyes from NP to proteins, which is otherwise not accessible with commonly used techniques of separation, such as dialysis and ultrafiltration/centrifugation employed in drug encapsulation and release studies of nanocarriers. Determining the interaction and transfer of dyes to proteins is of utmost importance in the pre-clinical evaluation of drug nanocarriers for improved correlation between in vitro and in vivo studies.
Assuntos
Proteínas Sanguíneas/análise , Portadores de Fármacos/química , Corantes Fluorescentes/química , Fracionamento por Campo e Fluxo/métodos , Nanopartículas/química , Polímeros/química , Adsorção , Fluorescência , Humanos , Hidrodinâmica , Cinética , Oxazinas/química , Teoria Quântica , Rodaminas/química , Espalhamento de RadiaçãoRESUMO
This article deals with the synthesis of Schiff-based bis-azomethine-based ligands derived from pyridoxal and aliphatic dihydrazides and the synthesis of nickel(II) complexes C1-C4. The synthesized complexes had their structures elucidated by monocrystal X-ray diffraction and were characterized by vibrational and absorption spectroscopy. The synthesized ligands have characteristics that allow the formation of self-assembly processes, thus, the flexibility or rigidity of the coordination of organic molecules added to the orbitals of the NiII cation leads to the formation of helical complexes with double helix and a dinucler nickel(II) complex. Moreover, compounds was their interactions with CT-DNA and HSA absorption and emission analysis and molecular docking calculations.