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1.
Bio Protoc ; 14(14): e5039, 2024 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-39100600

RESUMO

Peripheral membrane proteins (PMPs) are a subgroup of membrane-associated proteins that are water-soluble and bind to membranes, often reversibly, to perform their function. These proteins have been extensively studied in the aqueous state, but there is often a lack of high-resolution structural and functional studies of these proteins in the membrane-bound state. Currently, nuclear magnetic resonance (NMR) is among the best-equipped methods to study these relatively small proteins and domains, but current models have some disadvantages that prevent a full understanding of PMP interactions with membranes and lipids. Micelles, bicelles, and nanodiscs are all available for NMR observation but are based on synthetic lipids that may destabilize proteins or are too large to accommodate straightforward structural analysis. This protocol introduces a method for forming reverse micelles using lipids from natural sources, here called native reverse micelles. This technique allows the PMPs to embed within a shell of naturally derived lipids surrounding a small water core solubilized in an alkane solvent. PMP embedment in the lipid shell mimics binding to a cellular membrane. Here, naturally derived lipids from soy, bovine heart, and porcine brain are used in conjunction with n-dodecylphosphocholine (DPC) to encapsulate a PMP from either concentrated or dried protein, resulting in reverse micelles that may be confirmed via dynamic light scattering and NMR. This protocol allows for high-quality NMR data of PMPs interacting with membrane lipids within a biologically accurate environment. Key features • This protocol describes using natural lipids to construct reverse micelles for high-resolution NMR studies of proteins. • Initial optimization of encapsulation conditions proceeds through visual assessment, with dynamic light scattering (DLS) to measure size distribution, and NMR to observe protein behavior. • Membrane-interacting proteins are encapsulated in their membrane-bound state. Proteins that do not interact with membranes are housed in their water-solubilized state. • Structural, functional, and inhibitory studies may be performed on native reverse micelle-encapsulated proteins.

2.
Bio Protoc ; 14(15): e5045, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39131193

RESUMO

Membrane proteins play critical roles in cell physiology and pathology. The conventional way to study membrane proteins at protein levels is to use optimal detergents to extract proteins from membranes. Identification of the optimal detergent is tedious , and in some cases, the protein functions are compromised. While this detergent-based approach has produced meaningful results in membrane protein research, a lipid environment should be more suitable to recapture the protein's native folding and functions. This protocol describes how to prepare amphipathic membrane scaffold-proteins (MSPs)-based nanodiscs of a cation-coupled melibiose symporter of Salmonella enterica serovar Typhimurium (MelBSt), a member of the major facilitator superfamily. MSPs generate nano-assemblies containing membrane proteins surrounded by a patch of native lipids to better preserve their native conformations and functions. This protocol requires purified membrane protein in detergents, purified MSPs in solution, and detergent-destabilized phospholipids. The mixture of all three components at specific ratios is incubated in the presence of Bio-Beads SM-2 resins, which absorb all detergent molecules, allowing the membrane protein to associate with lipids surrounded by the MSPs. By reconstituting the purified membrane proteins back into their native-like lipid environment, these nanodisc-like particles can be directly used in cryo-EM single-particle analysis for structure determination and other biophysical analyses. It is noted that nanodiscs may potentially limit the dynamics of membrane proteins due to suboptimal nanodisc size compared to the native lipid bilayer. Key features • This protocol was built based on the method originally developed by Sligar et al. [1] and modified for a specific major facilitator superfamily transporter • This protocol is robust and reproducible • Lipid nanodiscs can increase membrane protein stability, and reconstituted transporters in lipid nanodiscs can regain function if their function is compromised using detergents • The reconstituted lipids nanodisc can be used for cryo-EM single-particle analysis.

3.
Nano Lett ; 24(32): 9808-9815, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39089683

RESUMO

Static electric fields play a considerable role in a variety of molecular nanosystems as diverse as single-molecule junctions, molecules supporting electrostatic catalysis, and biological cell membranes incorporating proteins. External electric fields can be applied to nanoscale samples with a conductive atomic force microscopy (AFM) probe in contact mode, but typically, no structural information is retrieved. Here we combine photothermal expansion infrared (IR) nanospectroscopy with electrostatic AFM probes to measure nanometric volumes where the IR field enhancement and the static electric field overlap spatially. We leverage the vibrational Stark effect in the polymer poly(methyl methacrylate) for calibrating the local electric field strength. In the relevant case of membrane protein bacteriorhodopsin, we observe electric-field-induced changes of the protein backbone conformation and residue protonation state. The proposed technique also has the potential to measure DC currents and IR spectra simultaneously, insofar enabling the monitoring of the possible interplay between charge transport and other effects.

4.
Crit Rev Food Sci Nutr ; : 1-22, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39106211

RESUMO

Milk fat globule membrane (MFGM) is a three-layer membrane-like structure encasing natural milk fat globules (MFGs). MFGM holds promise as a nutritional supplement because of the numerous physiological functions of its constituent protein. This review summarizes and compares the differences in MFGM protein composition across various species, including bovines, goats, camels, mares, and donkeys, and different lactation periods, such as colostrum and mature milk, as assessed by techniques such as proteomics and mass spectrometry. We also discuss the health benefits of MFGM proteins throughout life. MFGM proteins promote intestinal development, neurodevelopment, and glucose and lipid metabolism by upregulating tight junction protein expression, brain function-related genes, and glucose and fatty acid biosynthesis processes. We focus on the mechanisms underlying these beneficial effects of MFGM proteins. MFGM proteins activate key substances in in signaling pathways, such as the phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, and myosin light chain kinase signaling pathways. Overall, the consumption of MFGM proteins plays an essential role in conferring health benefits, some of which are important throughout the mammalian life cycle.


Types and amounts of MFGM proteins in mammals, as assessed by proteomic and mass spectrometry analysis, are summarized.Colostrum MFGM contains more acute phase proteins, whereas mature milk has higher levels of mucins (1 and 15), ADPH, XDH, and FABP.Health benefits of MFGM proteins, including intestinal development, neurodevelopment, and immune activity enhancement, are summarized.MFGM proteins have been shown to significantly activate the PI3K/Akt/mTOR signaling pathway, promoting cell proliferation and glycolipid metabolism.

5.
Int J Biol Macromol ; 278(Pt 1): 134219, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39097041

RESUMO

Cholesterol is a major component of plasma membranes and plays a significant role in actively regulating the functioning of several membrane proteins in humans. In this study, we focus on the role of cholesterol depletion on the voltage-gated sodium channel Nav1.7, which is primarily expressed in the peripheral sensory neurons and linked to various chronic inherited pain syndromes. Coarse-grained molecular dynamics simulations revealed key dynamic changes of Nav1.7 upon membrane cholesterol depletion: A loss of rigidity in the structural motifs linked to activation and fast-inactivation is observed, suggesting an easier transition of the channel between different gating states. In-vitro whole-cell patch clamp experiments on HEK293t cells expressing Nav1.7 validated these predictions at the functional level: Hyperpolarizing shifts in the voltage-dependence of activation and fast-inactivation were observed along with an acceleration of the time to peak and onset kinetics of fast inactivation. These results underline the critical role of membrane composition, and of cholesterol in particular, in influencing Nav1.7 gating characteristics. Furthermore, our results also point to cholesterol-driven changes of the geometry of drug-binding regions, hinting to a key role of the membrane environment in the regulation of drug effects.

6.
Drug Discov Today ; 29(9): 104130, 2024 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-39103143

RESUMO

Prostate cancer (PCa) is one of the leading cancers in men and the lack of suitable biomarkers or their modulators results in poor prognosis. Membrane proteins (MPs) have a crucial role in the development and progression of PCa and can be attractive therapeutic targets. However, experimental limitations in targeting MPs hinder effective biomarker and inhibitor discovery. To overcome this barrier, computational methods can yield structural insights and screen large libraries of compounds, accelerating lead identification and optimization. In this review, we examine current breakthroughs in computer-aided drug design (CADD), with emphasis on structure-based approaches targeting the most relevant membrane-bound PCa biomarkers.

7.
Methods Mol Biol ; 2843: 195-216, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39141302

RESUMO

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs.


Assuntos
Antígenos de Bactérias , Biotinilação , Vesículas Extracelulares , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Vacinas Bacterianas/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Desenvolvimento de Vacinas , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/imunologia
8.
Front Immunol ; 15: 1408415, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39148736

RESUMO

Exosomes play a crucial role in various biological processes, such as human development, immune responses, and disease occurrence. The membrane proteins on exosomes are pivotal factors for their biological functionality. Currently, numerous membrane proteins have been identified on exosome membranes, participating in intercellular communication, mediating target cell recognition, and regulating immune processes. Furthermore, membrane proteins from exosomes derived from cancer cells can serve as relevant biomarkers for early cancer diagnosis. This article provides a comprehensive review of the composition of exosome membrane proteins and their diverse functions in the organism's biological processes. Through in-depth exploration of exosome membrane proteins, it is expected to offer essential foundations for the future development of novel biomedical diagnostics and therapies.


Assuntos
Exossomos , Proteínas de Membrana , Exossomos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Animais , Neoplasias/imunologia , Neoplasias/metabolismo , Comunicação Celular , Biomarcadores Tumorais/metabolismo , Biomarcadores
9.
Methods Mol Biol ; 2780: 203-255, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38987471

RESUMO

Despite the recent advances in the determination of high-resolution membrane protein (MP) structures, the structural and functional characterization of MPs remains extremely challenging, mainly due to the hydrophobic nature, low abundance, poor expression, purification, and crystallization difficulties associated with MPs. Whereby the major challenges/hurdles for MP structure determination are associated with the expression, purification, and crystallization procedures. Although there have been significant advances in the experimental determination of MP structures, only a limited number of MP structures (approximately less than 1% of all) are available in the Protein Data Bank (PDB). Therefore, the structures of a large number of MPs still remain unresolved, which leads to the availability of widely unplumbed structural and functional information related to MPs. As a result, recent developments in the drug discovery realm and the significant biological contemplation have led to the development of several novel, low-cost, and time-efficient computational methods that overcome the limitations of experimental approaches, supplement experiments, and provide alternatives for the characterization of MPs. Whereby the fine tuning and optimizations of these computational approaches remains an ongoing endeavor.Computational methods offer a potential way for the elucidation of structural features and the augmentation of currently available MP information. However, the use of computational modeling can be extremely challenging for MPs mainly due to insufficient knowledge of (or gaps in) atomic structures of MPs. Despite the availability of numerous in silico methods for 3D structure determination the applicability of these methods to MPs remains relatively low since all methods are not well-suited or adequate for MPs. However, sophisticated methods for MP structure predictions are constantly being developed and updated to integrate the modifications required for MPs. Currently, different computational methods for (1) MP structure prediction, (2) stability analysis of MPs through molecular dynamics simulations, (3) modeling of MP complexes through docking, (4) prediction of interactions between MPs, and (5) MP interactions with its soluble partner are extensively used. Towards this end, MP docking is widely used. It is notable that the MP docking methods yet few in number might show greater potential in terms of filling the knowledge gap. In this chapter, MP docking methods and associated challenges have been reviewed to improve the applicability, accuracy, and the ability to model macromolecular complexes.


Assuntos
Bases de Dados de Proteínas , Proteínas de Membrana , Simulação de Acoplamento Molecular , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular/métodos , Ligação Proteica , Conformação Proteica , Biologia Computacional/métodos
10.
Biophys Chem ; 313: 107290, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-39002246

RESUMO

Due to their fundamental biological importance, membrane proteins (MPs) are attractive targets for drug discovery, with cell surface receptors, transporters, ion channels, and membrane-bound enzymes being of particular interest. However, due to numerous challenges, these proteins present underutilized opportunities for discovering biotherapeutics. Antibodies hold the promise of exquisite specificity and adaptability, making them the ideal candidates for targeting complex membrane proteins. They can target specific conformations of a particular membrane protein and can be engineered into various formats. Generating specific and effective antibodies targeting these proteins is no easy task due to several factors. The antigen's design, antibody-generation strategies, lead optimization technologies, and antibody modalities can be modified to tackle these challenges. The rational employment of cutting-edge lipid nanoparticle systems for retrieving the membrane antigen has been successfully implemented to simplify the mechanism-based therapeutic antibody discovery approach. Despite the highlighted MP production challenges, this review unequivocally underscores the advantages of targeting complex membrane proteins with antibodies and designing membrane protein antigens. Selected examples of lipid nanoparticle success have been illustrated, emphasizing the potential of therapeutic antibody discovery in this regard. With further research and development, we can overcome these challenges and unlock the full potential of therapeutic antibodies directed to target complex MPs.

11.
Int J Mol Sci ; 25(13)2024 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-39000557

RESUMO

The effects of intestinal microflora on extraintestinal immune response by intestinal cytokines and metabolites have been documented, but whether intestinal microbes stimulate serum antibody generation is unknown. Here, serum antibodies against 69 outer membrane proteins of Escherichia coli, a dominant bacterium in the human intestine, are detected in 141 healthy individuals of varying ages. Antibodies against E. coli outer membrane proteins are determined in all serum samples tested, and frequencies of antibodies to five outer membrane proteins (OmpA, OmpX, TsX, HlpA, and FepA) are close to 100%. Serum antibodies against E. coli outer membrane proteins are further validated by Western blot and bacterial pull-down. Moreover, the present study shows that OstA, HlpA, Tsx, NlpB, OmpC, YfcU, and OmpA provide specific immune protection against pathogenic E. coli, while HlpA and OmpA also exhibit cross-protection against Staphylococcus aureus infection. These finding indicate that intestinal E. coli activate extraintestinal antibody responses and provide anti-infective immunity.


Assuntos
Anticorpos Antibacterianos , Proteínas da Membrana Bacteriana Externa , Escherichia coli , Humanos , Escherichia coli/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Proteínas da Membrana Bacteriana Externa/imunologia , Adulto , Feminino , Staphylococcus aureus/imunologia , Masculino , Formação de Anticorpos/imunologia , Pessoa de Meia-Idade , Proteínas de Escherichia coli/imunologia , Adulto Jovem , Idoso , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Adolescente , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia
12.
Sci Rep ; 14(1): 17033, 2024 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-39043862

RESUMO

Tritrichomonas foetus is a flagellated and anaerobic parasite able to infect cattle and felines. Despite its prevalence, there is no effective standardized or legal treatment for T. foetus-infected cattle; the vaccination still has limited success in mitigating infections and reducing abortion risk; and nowadays, the diagnosis of T. foetus presents important limitations in terms of sensitivity and specificity in bovines. Here, we characterize the plasma membrane proteome of T. foetus and identify proteins that are represented in different isolates of this protozoan. Additionally, we performed a bioinformatic analysis that revealed the antigenicity potential of some of those proteins. This analysis is the first study to identify common proteins at the plasma membrane of different T. foetus isolates that could be targets for alternative diagnostic or vaccine techniques in the future.


Assuntos
Proteômica , Proteínas de Protozoários , Tritrichomonas foetus , Tritrichomonas foetus/isolamento & purificação , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/análise , Animais , Proteoma/análise , Membrana Celular/metabolismo , Bovinos , Proteínas de Membrana/metabolismo , Doenças dos Bovinos/parasitologia , Infecções Protozoárias em Animais/parasitologia , Infecções Protozoárias em Animais/diagnóstico , Biologia Computacional/métodos
13.
Methods Enzymol ; 701: 123-156, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39025570

RESUMO

Membrane proteins (MPs) often show preference for one phase over the other, which is characterized by the partition coefficient, Kp. The physical mechanisms underlying Kp have been only inferred indirectly from experiments due to the unavailability of detailed structures and compositions of ordered phases. Molecular dynamics (MD) simulations can complement these details and thus, in principle, provide further insights into the partitioning of MPs between two phases. However, the application of MD has remained difficult due to long time scales required for equilibration and large system size for the phase stability, which have not been fully resolved even in free energy simulations. This chapter describes the recently developed binary bilayer simulation method, where the membrane is composed of two laterally attached membrane patches. The binary bilayer system (BBS) is designed to preserve the lateral packing of both phases in a significantly smaller size compared to that required for macroscopic phase separation. These characteristics are advantageous in partitioning simulations, as the length scale for diffusion across the system can be significantly smaller. Hence the BBS can be efficiently employed in both conventional MD and free energy simulations, though sampling in ordered phases remains difficult due to slow diffusion. Development of efficient lipid swapping methods and its combination with the BBS would be a useful approach for partitioning in coexisting phases.


Assuntos
Bicamadas Lipídicas , Proteínas de Membrana , Simulação de Dinâmica Molecular , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Difusão , Termodinâmica
14.
Annu Rev Biophys ; 53(1): 427-453, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39013028

RESUMO

Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas de Membrana , Imagem Individual de Molécula , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Humanos , Animais
15.
Microbiol Spectr ; 12(8): e0415223, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-39012110

RESUMO

Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.


Assuntos
Proteínas de Bactérias , NAD , Quinona Redutases , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismo , NAD/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Quinona Redutases/metabolismo , Quinona Redutases/genética , NADP/metabolismo , Metabolismo Energético , Oxirredução
16.
Chempluschem ; : e202400340, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-39031638

RESUMO

Native mass spectrometry of membrane proteins relies on non-ionic detergents which protect the protein during transfer from solution into the gas phase. Once in the gas phase, the detergent micelle must be efficiently removed, which is usually achieved by collision-induced dissociation (CID). Recently, infrared multiple photon dissociation (IRMPD) has emerged as an alternative activation method for the analysis of membrane proteins, which has led to a growing interest in detergents that efficiently absorb infrared light. Here we investigate whether the absorption properties of synthetic detergents can be tailored by merging structural motifs of existing detergents into new hybrid detergents. We combine gas-phase infrared ion spectroscopy with density functional theory to investigate and rationalize the absorption properties of three established detergents and two hybrid detergents with fused headgroups. We show that, although the basic intramolecular interactions in the parent and hybrid detergents are similar, the three-dimensional structures differ significantly and so do the infrared spectra. Our results outline a roadmap for guiding the synthesis of tailored detergents with computational chemistry for future mass spectrometry applications.

17.
Methods Mol Biol ; 2814: 133-147, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954203

RESUMO

Activation processes at the plasma membrane have been studied with life-cell imaging using GFP fused to a protein that binds to a component of the activation process. In this way, PIP3 formation has been monitored with CRAC-GFP, Ras-GTP with RBD-Raf-GFP, and Rap-GTP with Ral-GDS-GFP. The fluorescent sensors translocate from the cytoplasm to the plasma membrane upon activation of the process. Although this translocation assay can provide very impressive images and movies, the method is not very sensitive, and amount of GFP-sensor at the plasma membrane is not linear with the amount of activator. The fluorescence in pixels at the cell boundary is partly coming from the GFP-sensor that is bound to the activated membrane and partly from unbound GFP-sensor in the cytosolic volume of that boundary pixel. The variable and unknown amount of cytosol in boundary pixels causes the low sensitivity and nonlinearity of the GFP-translocation assay. Here we describe a method in which the GFP-sensor is co-expressed with cytosolic-RFP. For each boundary pixels, the RFP fluorescence is used to determine the amount of cytosol of that pixel and is subtracted from the GFP fluorescence of that pixel yielding the amount of GFP-sensor that is specifically associated with the plasma membrane in that pixel. This GRminusRD method using GFP-sensor/RFP is at least tenfold more sensitive, more reproducible, and linear with activator compared to GFP-sensor alone.


Assuntos
Membrana Celular , Proteínas de Fluorescência Verde , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Luminescentes/metabolismo , Proteínas Luminescentes/genética , Transporte Proteico , Microscopia de Fluorescência/métodos , Citosol/metabolismo , Animais
18.
Sci Rep ; 14(1): 15992, 2024 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987432

RESUMO

Aquaporins (AQPs) are a family of water permeable channels expressed on the plasma membrane with AQP5 being the major channel expressed in several human tissues including salivary and lacrimal glands. Anti-AQP5 autoantibodies have been observed in patients with Sjögren's syndrome who are characterised by dryness of both salivary and lacrimal glands, and they have been implicated in the underlying mechanisms of glandular dysfunction. AQP5 is formed by six transmembrane helices linked with three extracellular and two intracellular loops. Develop antibodies against membrane protein extracellular loops can be a challenge due to the difficulty in maintaining these proteins as recombinant in their native form. Therefore, in this work we aimed to generate an efficient stable-transfected cell line overexpressing human AQP5 (CHO-K1/AQP5) to perform primarily cell-based phage display biopanning experiments to develop new potential recombinant antibodies targeting AQP5. We also showed that the new CHO-K1/AQP5 cell line can be used to study molecular mechanisms of AQP5 sub-cellular trafficking making these cells a useful tool for functional studies.


Assuntos
Aquaporina 5 , Cricetulus , Aquaporina 5/metabolismo , Aquaporina 5/genética , Células CHO , Humanos , Animais , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Anticorpos/metabolismo , Biblioteca de Peptídeos
19.
Molecules ; 29(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38930903

RESUMO

A method is described to deconstruct the network of hydropathic interactions within and between a protein's sidechain and its environment into residue-based three-dimensional maps. These maps encode favorable and unfavorable hydrophobic and polar interactions, in terms of spatial positions for optimal interactions, relative interaction strength, as well as character. In addition, these maps are backbone angle-dependent. After map calculation and clustering, a finite number of unique residue sidechain interaction maps exist for each backbone conformation, with the number related to the residue's size and interaction complexity. Structures for soluble proteins (~749,000 residues) and membrane proteins (~387,000 residues) were analyzed, with the latter group being subdivided into three subsets related to the residue's position in the membrane protein: soluble domain, core-facing transmembrane domain, and lipid-facing transmembrane domain. This work suggests that maps representing residue types and their backbone conformation can be reassembled to optimize the medium-to-high resolution details of a protein structure. In particular, the information encoded in maps constructed from the lipid-facing transmembrane residues appears to paint a clear picture of the protein-lipid interactions that are difficult to obtain experimentally.


Assuntos
Proteínas de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica , Lipídeos/química , Ligação Proteica
20.
Int J Mol Sci ; 25(12)2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38928089

RESUMO

SARS-CoV-2 S-protein-mediated fusion is thought to involve the interaction of the membrane-distal or N-terminal heptad repeat (NHR) ("HR1") of the cleaved S2 segment of the protein and the membrane-proximal or C-terminal heptad repeat (CHR) ("HR2") regions of the protein. We examined the fusion inhibitory activity of a PEGylated HR2-derived peptide and its palmitoylated derivative using a pseudovirus infection assay. The latter peptide caused a 76% reduction in fusion activity at 10 µM. Our results suggest that small variations in peptide derivatization and differences in the membrane composition of pseudovirus preparations may affect the inhibitory potency of HR2-derived peptides. We suggest that future studies on the inhibition of infectivity of SARS-CoV-2 in both in vitro and in vivo systems consider the need for higher concentrations of peptide inhibitors.


Assuntos
Peptídeos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Peptídeos/farmacologia , Peptídeos/química , Ácido Palmítico/farmacologia , Ácido Palmítico/química , Internalização do Vírus/efeitos dos fármacos , COVID-19/virologia , COVID-19/metabolismo , Antivirais/farmacologia , Antivirais/química
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