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1.
Indian J Public Health ; 68(2): 302-304, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38953823

RESUMO

Knowledge of the consequences of uncontrolled diabetes mellitus and hypertension on various body organs among health workers is necessary to educate patients. Body mapping is a tool used for exploring perceptions as a part of qualitative research. This study assesses the perceptions of health-care workers on the effects of uncontrolled diabetes mellitus and hypertension on the human body using the body mapping technique. All 19 staff members of an Urban Primary Health Center were asked to draw a human body and map the parts affected by uncontrolled diabetes mellitus and hypertension. The mean age of the participants was 35.42 ± 10.54 years, and median years of work experience were 4 (3, 7) years. Fourteen (74%) participants had mapped kidneys and 11 (58%) participants had mapped head/brain indicating stroke to indicate damage due to uncontrolled diabetes mellitus and hypertension. Only 7 (37%) and 4 (26%) participants perceived that feet and eyes could be affected.


Assuntos
Diabetes Mellitus , Pessoal de Saúde , Hipertensão , Humanos , Hipertensão/psicologia , Adulto , Masculino , Feminino , Diabetes Mellitus/psicologia , Pessoal de Saúde/psicologia , Pessoa de Meia-Idade , Percepção , Atitude do Pessoal de Saúde , Índia , Conhecimentos, Atitudes e Prática em Saúde
2.
Nanoscale ; 16(28): 13503-13515, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-38940744

RESUMO

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain ß-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.


Assuntos
Bicamadas Lipídicas , Proteínas de Membrana , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Técnicas de Microbalança de Cristal de Quartzo
3.
EMBO Rep ; 25(3): 1310-1325, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38321165

RESUMO

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.


Assuntos
Poxviridae , Vacínia , Humanos , Sulfatos de Condroitina , Vaccinia virus/metabolismo , Poxviridae/metabolismo , Proteínas Virais/metabolismo , Fusão de Membrana , Proteínas de Transporte
4.
EMBO Rep ; 25(1): 82-101, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38228789

RESUMO

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Membrana/metabolismo , Transporte Biológico , Lipoproteínas/metabolismo
5.
Biochem Biophys Rep ; 37: 101636, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38283190

RESUMO

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene) type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with a UBE2 (ubiquitin conjugating enzyme). Our understanding of MuRF1 function remains unclear as candidate UBE2s have not been fully elucidated. In the present study, we screened human ubiquitin dependent UBE2s in vitro and found that MuRF1 engages in ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. MuRF1 can cause mono-ubiquitylation, K48- and K63-linked polyubiquitin chains in a UBE2 dependent manner. Moreover, we identified a two-step UBE2 dependent mechanism whereby MuRF1 is monoubiquitylated by UBE2W which acts as an anchor for UBE2N/V to generate polyubiquitin chains. With the in vitro ubiquitylation assay, we also found that MuRF2 and MuRF3 not only share the same UBE2 partners as MuRF1 but can also directly ubiquitylate the same substrates: Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). In summary, our work presents new insights into the mechanisms that underpin MuRF1 activity and reveals overlap in MuRF-induced ubiquitylation which could explain their partial redundancy in vivo.

6.
Methods Mol Biol ; 2659: 61-71, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37249885

RESUMO

Chemotropism refers to the directional growth of a living organism toward a chemical stimulus. Molecular mechanisms underlying chemotropism of fungal pathogens have recently been enabled by advancements in biological chemotropic assays, with a particular focus on the roles of G-protein-coupled receptors and their plant-derived ligands in chemotropism. Here we describe in detail an assay that enables quantification of chemotropic responses of Fusarium graminearum, with variations recently reported for Fusarium oxysporum and Trichoderma atroviride.


Assuntos
Quimiotaxia , Fusarium , Plantas , Receptores Acoplados a Proteínas G , Doenças das Plantas/microbiologia
7.
Indian J Community Med ; 48(6): 937-938, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38249703
8.
Front Cell Infect Microbiol ; 13: 1287418, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38239502

RESUMO

Introduction: The fungal G protein-coupled receptors Ste2 and Ste3 are vital in mediating directional hyphal growth of the agricultural pathogen Fusarium graminearum towards wheat plants. This chemotropism is induced by a catalytic product of peroxidases secreted by the wheat. Currently, the identity of this product, and the substrate it is generated from, are not known. Methods and results: We provide evidence that a peroxidase substrate is derived from F. graminearum conidia and report a simple method to extract and purify the FgSte2-activating ligand for analyses by mass spectrometry. The mass spectra arising from t he ligand extract are characteristic of a 400 Da carbohydrate moiety. Consistent with this type of molecule, glycosidase treatment of F. graminearum conidia prior to peroxidase treatment significantly reduced the amount of ligand extracted. Interestingly, availability of the peroxidase substrate appears to depend on the presence of both FgSte2 and FgSte3, as knockout of one or the other reduces the chemotropism-inducing effect of the extracts. Conclusions: While further characterization is necessary, identification of the F. graminearum-derived peroxidase substrate and the FgSte2-activating ligand will unearth deeper insights into the intricate mechanisms that underlie fungal pathogenesis in cereal crops, unveiling novel avenues for inhibitory interventions.


Assuntos
Fusarium , Peroxidase , Ligantes , Peroxidases/farmacologia , Doenças das Plantas/microbiologia
9.
mSphere ; 7(6): e0045622, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36377914

RESUMO

Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Previous studies on Fusarium species have highlighted the involvement of the Ste2 G-protein-coupled receptor (GPCR) in mediating polarized hyphal growth toward host-released peroxidase. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to Fusarium graminearum chemotropism and pathogenicity. Fgste3Δ deletion strains were found to be compromised in the chemotropic response toward peroxidase, development of lesions on germinating wheat, and infection of Arabidopsis thaliana leaves. In the absence of FgSte3 or FgSte2, F. graminearum cells exposed to peroxidase showed no phosphorylation of the cell-wall integrity, mitogen-activated protein kinase pathway component Mgv1. In addition, transcriptomic gene expression profiling yielded a list of genes involved in cellular reorganization, cell wall remodeling, and infection-mediated responses that were differentially modulated by peroxidase when FgSte3 was present. Deletion of FgSte3 yielded the downregulation of genes associated with mycotoxin biosynthesis and appressorium development, compared to the wild-type strain, both in the presence of peroxidase. Together, these findings contribute to our understanding of the mechanism underlying fungal chemotropism and pathogenesis while raising the novel hypothesis that FgSte2 and FgSte3 are interdependent on each other for the mediation of the redirection of hyphal growth in response to host-derived peroxidase. IMPORTANCE Fusarium head blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to F. graminearum chemotropism and pathogenicity. These findings contribute to our understanding of the mechanisms underlying fungal chemotropism and pathogenesis.


Assuntos
Fusarium , Fusarium/genética , Peroxidases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peroxidase , Receptores Acoplados a Proteínas G/metabolismo
10.
Nature ; 606(7916): 953-959, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35705811

RESUMO

Linkages between the outer membrane of Gram-negative bacteria and the peptidoglycan layer are crucial for the maintenance of cellular integrity and enable survival in challenging environments1-5. The function of the outer membrane is dependent on outer membrane proteins (OMPs), which are inserted into the membrane by the ß-barrel assembly machine6,7 (BAM). Growing Escherichia coli cells segregate old OMPs towards the poles by a process known as binary partitioning, the basis of which is unknown8. Here we demonstrate that peptidoglycan underpins the spatiotemporal organization of OMPs. Mature, tetrapeptide-rich peptidoglycan binds to BAM components and suppresses OMP foldase activity. Nascent peptidoglycan, which is enriched in pentapeptides and concentrated at septa9, associates with BAM poorly and has little effect on its activity, leading to preferential insertion of OMPs at division sites. The synchronization of OMP biogenesis with cell wall growth results in the binary partitioning of OMPs as cells divide. Our study reveals that Gram-negative bacteria coordinate the assembly of two major cell envelope layers by rendering OMP biogenesis responsive to peptidoglycan maturation, a potential vulnerability that could be exploited in future antibiotic design.


Assuntos
Proteínas da Membrana Bacteriana Externa , Membrana Celular , Escherichia coli , Peptidoglicano , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Peptidoglicano/metabolismo , Dobramento de Proteína
11.
Biophys J ; 120(23): 5295-5308, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34757080

RESUMO

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the ß-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring, we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin, and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.


Assuntos
Proteínas de Escherichia coli , Proteínas da Membrana Bacteriana Externa , Escherichia coli , Dobramento de Proteína
12.
Nanoscale ; 13(31): 13519-13528, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477756

RESUMO

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N',N'-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a 'toolkit' of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.


Assuntos
Proteínas de Membrana , Polímeros , Bicamadas Lipídicas , Maleatos , Poliestirenos , Rodopsina , Estireno
13.
Elife ; 92020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33315009

RESUMO

The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell division site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Transporte Proteico/fisiologia , Antibacterianos/metabolismo , Parede Celular/metabolismo , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Lipoproteínas/metabolismo , Fatores de Virulência/metabolismo
14.
Sci Rep ; 10(1): 10770, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32612109

RESUMO

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum 'Roblin' cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.


Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Receptores de Fator de Acasalamento/metabolismo , Triticum/microbiologia , Agrobacterium tumefaciens , Catálise , Parede Celular/metabolismo , Quimiotaxia , Deleção de Genes , Hifas/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases , Peroxidases/metabolismo , Feromônios/metabolismo , Doenças das Plantas/microbiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais , Esporos Fúngicos/metabolismo , Virulência
15.
Commun Biol ; 3(1): 83, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081916

RESUMO

Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.


Assuntos
Plaquinas/química , Plaquinas/metabolismo , Vimentina/química , Vimentina/metabolismo , Sequência de Aminoácidos , Aminoácidos Acídicos/química , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Células HeLa , Humanos , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Plaquinas/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Vimentina/genética
16.
Nat Microbiol ; 4(10): 1692-1705, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31235958

RESUMO

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting ß-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Cristalografia por Raios X , Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Periplasma/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta
17.
Biochim Biophys Acta Biomembr ; 1861(8): 1437-1445, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31150633

RESUMO

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.


Assuntos
Proteínas de Membrana/química , Nanotecnologia , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Western Blotting , Lipídeos/química , Espectrometria de Massas , Microscopia Eletrônica , Estrutura Quaternária de Proteína
18.
Nanoscale ; 10(22): 10609-10619, 2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29845165

RESUMO

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form "SMA Lipid Particles" (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. "SMILPs" thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.


Assuntos
Bicamadas Lipídicas/química , Maleatos/química , Nanopartículas/química , Poliestirenos/química , Células HEK293 , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Solubilidade
19.
ACS Chem Biol ; 12(10): 2631-2643, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28862425

RESUMO

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag's are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.


Assuntos
Antígenos CD/metabolismo , Butirofilinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Antígenos , Antígenos CD/genética , Butirofilinas/genética , Clonagem Molecular , Técnicas de Cocultura , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mutação , Fosfoproteínas , Conformação Proteica , Domínios Proteicos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/metabolismo
20.
Nat Protoc ; 11(7): 1149-62, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27254461

RESUMO

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.


Assuntos
Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/química , Maleatos/química , Lipídeos de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Poliestirenos/química , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Solubilidade
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