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OBJECTIVES: Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive. METHODS: We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment. RESULTS: The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity. CONCLUSIONS: These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion.
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Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-ß 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.
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Aplysia , Espectrometria de Massas , Receptores Nicotínicos , Animais , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/química , Espectrometria de Massas/métodos , Sítios de Ligação , Ligação Proteica/fisiologia , Proteínas de Transporte/metabolismo , Bungarotoxinas/farmacologia , Bungarotoxinas/metabolismo , Bungarotoxinas/química , Acetilcolina/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Modelos MolecularesRESUMO
Human babesiosis is a rapidly emerging and potentially fatal tick-borne disease caused by intraerythrocytic apicomplexan parasites of the Babesia genus. Among the various species of Babesia that infect humans, B. duncani has been found to cause severe and life-threatening infections. Detection of active B. duncani infection is critical for accurate diagnosis and effective management of the disease. While molecular assays for the detection of B. duncani infection in blood are available, a reliable strategy to detect biomarkers of active infection has not yet been developed. Here, we report the development of the first B. duncani antigen capture assays that rely on the detection of two B. duncani -exported immunodominant antigens, BdV234 and BdV38. The assays were validated using blood samples from cultured parasites in human erythrocytes and B. duncani -infected laboratory mice at different parasitemia levels and following therapy. The assays display high specificity with no cross-reactivity with B. microti , B. divergens , Babesia MO1, or P. falciparum. The assay also demonstrates high sensitivity, detecting as low as 115 infected erythrocytes/µl of blood. Screening of 1,731 blood samples from diverse biorepositories, including previously identified Lyme and/or B. microti positive human samples and new specimens from field mice, showed no evidence of B. duncani infection in these samples. The assays could be useful in diverse diagnostic scenarios, including point-of-care testing for early B. duncani infection detection in patients, field tests for screening reservoir hosts, and high-throughput screening such as blood collected for transfusion. Short summary: We developed two ELISA-based assays, BdACA38 and BdACA234, for detecting B. duncani , a potentially fatal tick-borne parasite causing human babesiosis. The assays target two immunodominant antigens, BdV234 and BdV38, demonstrating high specificity (no cross-reactivity with other Babesia species or Plasmodium falciparum ) and sensitivity (detecting as low as 115 infected erythrocytes/µl). The assays were validated using in vitro-cultured parasites and infected mice. Screening diverse blood samples showed no evidence of B. duncani active infection among 1,731 human and field mice blood samples collected from the north-eastern, midwestern, and western US. These assays offer potential in diverse diagnostic scenarios, including early patient detection, reservoir animal screening, and transfusion-transmitted babesiosis prevention.
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Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).
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Adesivos , Farinha , Glutens , Proteoma , Amido , Triticum , Triticum/química , Farinha/análise , Amido/química , Proteoma/análise , Proteoma/química , Adesivos/química , Glutens/química , Glutens/análise , Proteômica/métodos , Proteínas de Plantas/análise , Gliadina/química , Gliadina/análiseRESUMO
The combination of advanced mass spectrometry and enrichment-based sample preparation methods has enhanced analytical capabilities in clinical proteomics. In this chapter, we describe a method of proteome analysis to identify Borrelia-derived peptides in urine that includes a sample affinity enrichment method coupled with liquid chromatography tandem mass spectrometry analysis and a bioinformatic peptide authentication algorithm.
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Borrelia , Humanos , Espectrometria de Massas em Tandem/métodos , Peptídeos/química , Cromatografia Líquida/métodos , Proteoma/análiseRESUMO
Chemotherapy resistance is considered one of the main causes of tumor relapse, still challenging researchers for the identification of the molecular mechanisms sustaining its emergence. Here, we setup and characterized chemotherapy-resistant models of Medulloblastoma (MB), one of the most lethal pediatric brain tumors, to uncover targetable vulnerabilities associated to their resistant phenotype. Integration of proteomic, transcriptomic and kinomic data revealed a significant deregulation of several pathways in resistant MB cells, converging to cell metabolism, RNA/protein homeostasis, and immune response, eventually impacting on patient outcome. Moreover, resistant MB cell response to a large library of compounds through a high-throughput screening (HTS), highlighted nucleoside metabolism as a relevant vulnerability of chemotolerant cells, with peculiar antimetabolites demonstrating increased efficacy against them and even synergism with conventional chemotherapeutics. Our results suggest that drug-resistant cells significantly rewire multiple cellular processes, allowing their adaptation to a chemotoxic environment, nevertheless exposing alternative actionable susceptibilities for their specific targeting.
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Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Criança , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Nucleosídeos/farmacologia , Nucleosídeos/uso terapêutico , Proteômica , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Linhagem Celular TumoralRESUMO
The PD-1/PD-L1 checkpoint pathway is important for regulating immune responses and can be targeted by immunomodulatory drugs to treat a variety of immune disorders. However, the precise protein-protein interactions required for the initiation of PD-1/PD-L1 signaling are currently unknown. Previously, we designed a series of first-generation PD-1 targeting peptides based on the native interface region of programmed death ligand 1 (PD-L1) that effectively reduced PD-1/PD-L1 binding. In this work, we further characterized the previously identified lead peptide, MN1.1, to identify key PD-1 binding residues and design an optimized peptide, MN1.4. We show MN1.4 is significantly more stable than MN1.1 in serum and retains the ability to block PD-1/PD-L1 complex formation. We further characterized the immunomodulatory effects of MN1.4 treatment by measuring markers of T cell activation in a co-culture model with ovarian cancer cells and peripheral blood mononuclear cells. We found MN1.4 treatment reduced cytokine secretion and suppressed T cell responses in a similar manner as recombinant PD-L1. Therefore, the PD-L1 interface region used to design MN1.4 appeared sufficient to initiate PD-1 signaling and likely represents the minimum necessary region of PD-L1 required for PD-1 recognition. We propose a peptide agonist for PD-1, such as MN1.4, could have several applications for treating autoimmune disorders caused by PD-1 deficiencies such as type 1 diabetes, inflammatory arthritis, or autoimmune side effects arising from monoclonal antibody-based cancer immunotherapies.
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Antígeno B7-H1 , Modelos Moleculares , Neoplasias , Transdução de Sinais , Humanos , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Imunoterapia , Leucócitos Mononucleares/metabolismo , Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Receptor de Morte Celular Programada 1/agonistas , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Mutação , Estrutura Quaternária de Proteína , Linhagem Celular Tumoral , Imunidade/efeitos dos fármacosRESUMO
Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) enabled for a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. Pure GPL mixtures at significantly high yields were produced through this approach. For this study, we utilised phoshatidylcholine (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bilayers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very comparable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.
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Hidrogênio , Bicamadas Lipídicas , Bicamadas Lipídicas/química , Glicerofosfolipídeos , Espalhamento a Baixo Ângulo , Difração de Raios X , FosfatidilgliceróisRESUMO
With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.
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Sistemas de Liberação de Medicamentos , Proteína Acessória do Receptor de Interleucina-1 , Animais , Universidades , DNA , Descoberta de DrogasRESUMO
Different amphiphilic co-polymers have been introduced to produce polymer-lipid particles with nanodisc structure composed of an inner lipid bilayer and polymer chains self-assembled as an outer belt. These particles can be used to stabilize membrane proteins in solution and enable their characterization by means of biophysical methods, including small-angle X-ray scattering (SAXS). Some of these co-polymers have also been used to directly extract membrane proteins together with their associated lipids from native membranes. Styrene/maleic acid and diisobutylene/maleic acid are among the most commonly used co-polymers for producing polymer-lipid particles, named SMALPs and DIBMALPs, respectively. Recently, a new co-polymer, named Glyco-DIBMA, was produced by partial amidation of DIBMA with the amino sugar N-methyl-d-glucosamine. Polymer-lipid particles produced with Glyco-DIBMA, named Glyco-DIBMALPs, exhibit improved structural properties and stability compared to those of SMALPs and DIBMALPs while retaining the capability of directly extracting membrane proteins from native membranes. Here, we characterize the structure and lipid composition of Glyco-DIBMALPs produced with either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Glyco-DIBMALPs were also prepared with mixtures of either POPC or DMPC and cholesterol at different mole fractions. We estimated the lipid content in the Glyco-DIBMALPs and determined the particle structure and morphology by SAXS. We show that the Glyco-DIBMALPs are nanodisc-like particles whose size and shape depend on the polymer/lipid ratio. This is relevant for designing nanodisc particles with a tunable diameter according to the size of the membrane protein to be incorporated. We also report that the addition of >20 mol % cholesterol strongly perturbed the formation of Glyco-DIBMALPs. Altogether, we describe a detailed characterization of the Glyco-DIBMALPs, which provides relevant inputs for future application of these particles in the biophysical investigation of membrane proteins.
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Dimiristoilfosfatidilcolina , Bicamadas Lipídicas , Dimiristoilfosfatidilcolina/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Bicamadas Lipídicas/química , Maleatos/química , Polímeros/química , Proteínas de Membrana/química , Colesterol/químicaRESUMO
Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both α-helical and ß-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with α-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.
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We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.
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Vesículas Extracelulares , Neoplasias , Animais , Líquido Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Mitofagia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte VesicularRESUMO
Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs.
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Fator VIIa , Tromboplastina , Fator VIIa/química , Fator VIIa/metabolismo , Bicamadas Lipídicas/química , Modelos Estruturais , Simulação de Dinâmica Molecular , Tromboplastina/química , Tromboplastina/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S)-pseudotyped viruses are commonly used for quantifying antiviral drugs and neutralizing antibodies. Here, we describe the development of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirion, which is a non-replicating SARS-CoV-2 virus-like particle composed of viral structural proteins (S, M, N, and E) and an RNA genome derived from a fast-expressing alphaviral vector. We validated Ha-CoV-2 for rapid quantification of neutralization antibodies, antiviral drugs, and viral variants. In addition, as a proof of concept, we used Ha-CoV-2 to quantify the neutralizing antibodies from an infected and vaccinated individual and found that the one-dose vaccination with Moderna mRNA-1273 greatly increased the anti-serum titer by approximately 6-fold. The post-vaccination serum can neutralize all nine variants tested. These results demonstrate that Ha-CoV-2 can be used as a robust platform for the rapid quantification of neutralizing antibodies against SARS-CoV-2 and its emerging variants.
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Alphavirus , COVID-19 , Humanos , SARS-CoV-2/genética , Anticorpos Neutralizantes , Alphavirus/genética , Antivirais/farmacologiaRESUMO
SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
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COVID-19/virologia , Bicamadas Lipídicas/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , COVID-19/metabolismo , COVID-19/fisiopatologia , Linhagem Celular , Humanos , Fusão de Membrana/fisiologia , Difração de Nêutrons/métodos , Ligação Proteica , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Internalização do VírusRESUMO
The bacterial cytoplasmic membrane is the innermost bacterial membrane and is mainly composed of three different phospholipid species, i.e., phosphoethanolamine (PE), phosphoglycerol (PG), and cardiolipin (CL). In particular, PG and CL are responsible for the negative charge of the membrane and are often the targets of cationic antimicrobial agents. The growing resistance of bacteria toward the available antibiotics requires the development of new and more efficient antibacterial drugs. In this context, studying the physicochemical properties of the bacterial cytoplasmic membrane is pivotal for understanding drug-membrane interactions at the molecular level as well as for designing drug-testing platforms. Here, we discuss the preparation and characterization of PE/PG/CL vesicle suspensions, which contain all of the main lipid components of the bacterial cytoplasmic membrane. The vesicle suspensions were characterized by means of small-angle neutron scattering, dynamic light scattering, and electron paramagnetic spectroscopy. By combining solution scattering and spectroscopy techniques, we propose a detailed description of the impact of different CL concentrations on the structure and dynamics of the PE/PG bilayer. CL induces the formation of thicker bilayers, which exhibit higher curvature and are overall more fluid. The experimental results contribute to shed light on the structure and dynamics of relevant model systems of the bacterial cytoplasmic membrane.
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Cardiolipinas , Bicamadas Lipídicas , Bactérias , Membrana Celular , FosfatidiletanolaminasRESUMO
Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.
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Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.