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1.
Funct Integr Genomics ; 24(2): 47, 2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38430379

RESUMO

Amino acid transporters (AATs) are essential integral membrane proteins that serve multiple roles, such as facilitating the transport of amino acids across cell membranes. They play a crucial role in the growth and development of plants. Phaseolus vulgaris, a significant legume crop, serves as a valuable model for studying root symbiosis. In this study, we have conducted an exploration of the AAT gene family in P. vulgaris. In this research, we identified 84 AAT genes within the P. vulgaris genome sequence and categorized them into 12 subfamilies based on their similarity and phylogenetic relationships with AATs found in Arabidopsis and rice. Interestingly, these AAT genes were not evenly distributed across the chromosomes of P. vulgaris . Instead, there was an unusual concentration of these genes located toward the outer edges of chromosomal arms. Upon conducting motif analysis and gene structural analysis, we observed a consistent presence of similar motifs and an intron-exon distribution pattern among the subfamilies. When we analyzed the expression profiles of PvAAT genes, we noted tissue-specific expression patterns. Furthermore, our investigation into AAT gene expression under rhizobial and mycorrhizal symbiotic conditions revealed that certain genes exhibited high levels of expression. Specifically, ATLa5 and LHT2 was notably upregulated under both symbiotic conditions. These findings point towards a potential role of AATs in the context of rhizobial and mycorrhizal symbiosis in P. vulgaris, in addition to their well-established regulatory functions.


Assuntos
Arabidopsis , Phaseolus , Rhizobium , Simbiose/genética , Phaseolus/genética , Filogenia , Sistemas de Transporte de Aminoácidos/genética , Membrana Celular
2.
Food Microbiol ; 120: 104484, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38431329

RESUMO

Trichothecium roseum is a typical necrotrophic fungal pathogen that not only bring about postharvest disease, but contribute to trichothecenes contamination in fruit and vegetables. Phospholipase D (PLD), as an important membrane lipid degrading enzyme, can produce phosphatidic acid (PA) by hydrolyzing phosphatidylcholine (PC) and phosphatidylinositol (PI). PA can promote the production of reactive oxygen species (ROS) by activating the activity of NADPH oxidase (NOX), thereby increasing the pathogenicity to fruit. However, the ROS mediated by TrPLD3 how to influence T. roseum infection to fruit by modulating phosphatidic acid metabolism, which has not been reported. In this study, the knockout mutant and complement strain of TrPLD3 were constructed through homologous recombination, TrPLD3 was tested for its effect on the colony growth and pathogenicity of T. roseum. The experimental results showed that the knockout of TrPLD3 inhibited the colony growth of T. roseum, altered the mycelial morphology, completely inhibited the sporulation, and reduced the accumulation of T-2 toxin. Moreover, the knockout of TrPLD3 significantly decreased pathogenicity of T. roseum on apple fruit. Compared to inoculated apple fruit with the wide type (WT), the production of ROS in apple infected with ΔTrPLD3 was slowed down, the relative expression and enzymatic activity of NOX, and PA content decreased, and the enzymatic activity and gene expression of superoxide dismutase (SOD) increased. In addition, PLD, lipoxygenase (LOX) and lipase activities were considerably decreased in apple fruit infected with ΔTrPLD3, the changes of membrane lipid components were slowed down, the decrease of unsaturated fatty acid content was alleviated, and the accumulation of saturated fatty acid content was reduced, thereby maintaining the cell membrane integrity of the inoculated apple fruit. We speculated that the decreased PA accumulation in ΔTrPLD3-inoculated apple fruit further weakened the interaction between PA and NOX on fruit, resulting in the reduction of ROS accumulation of fruits, which decreased the damage to the cell membrane and maintained the cell membrane integrity, thus reducing the pathogenicity to apple. Therefore, TrPLD3-mediated ROS plays a critical regulatory role in reducing the pathogenicity of T. roseum on apple fruit by influencing phosphatidic acid metabolism.


Assuntos
Frutas , Hypocreales , Malus , Frutas/microbiologia , Malus/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo
3.
Rep Prog Phys ; 87(3)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38433567

RESUMO

This review examines the biological physics of intracellular transport probed by the coherent optics of dynamic light scattering from optically thick living tissues. Cells and their constituents are in constant motion, composed of a broad range of speeds spanning many orders of magnitude that reflect the wide array of functions and mechanisms that maintain cellular health. From the organelle scale of tens of nanometers and upward in size, the motion inside living tissue is actively driven rather than thermal, propelled by the hydrolysis of bioenergetic molecules and the forces of molecular motors. Active transport can mimic the random walks of thermal Brownian motion, but mean-squared displacements are far from thermal equilibrium and can display anomalous diffusion through Lévy or fractional Brownian walks. Despite the average isotropic three-dimensional environment of cells and tissues, active cellular or intracellular transport of single light-scattering objects is often pseudo-one-dimensional, for instance as organelle displacement persists along cytoskeletal tracks or as membranes displace along the normal to cell surfaces, albeit isotropically oriented in three dimensions. Coherent light scattering is a natural tool to characterize such tissue dynamics because persistent directed transport induces Doppler shifts in the scattered light. The many frequency-shifted partial waves from the complex and dynamic media interfere to produce dynamic speckle that reveals tissue-scale processes through speckle contrast imaging and fluctuation spectroscopy. Low-coherence interferometry, dynamic optical coherence tomography, diffusing-wave spectroscopy, diffuse-correlation spectroscopy, differential dynamic microscopy and digital holography offer coherent detection methods that shed light on intracellular processes. In health-care applications, altered states of cellular health and disease display altered cellular motions that imprint on the statistical fluctuations of the scattered light. For instance, the efficacy of medical therapeutics can be monitored by measuring the changes they induce in the Doppler spectra of livingex vivocancer biopsies.


Assuntos
Citoesqueleto , Membrana Celular , Movimento Celular , Transporte Biológico , Difusão Dinâmica da Luz
4.
PeerJ ; 12: e16790, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38436004

RESUMO

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.


Assuntos
Oryza , Oryza/genética , Parede Celular , Membrana Celular , Peroxidase , Álcalis
5.
Nat Commun ; 15(1): 1528, 2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453906

RESUMO

The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4's trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway.


Assuntos
Transdução de Sinais , Receptor 4 Toll-Like , Animais , Criança , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo , Lipopolissacarídeos/metabolismo , Receptor 4 Toll-Like/metabolismo
6.
Int J Nanomedicine ; 19: 2395-2407, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469059

RESUMO

Background: Recently, the use of the tumor or its secretions as drug carriers has gradually become popular, with the advantages of high biocompatibility and enhanced drug delivery to specific cells. Melanoma is the most malignant tumor of all skin cancers; it is the most metastatic and, therefore, the most difficult to treat. The main purpose of this study is to develop nanovesicles with tumor cell membrane secretion properties to encapsulate target substances to enhance the therapeutic effect of cancer. Methods: Astaxanthin was selected as an anticancer drug due to our previous research finding that astaxanthin has extremely high antioxidant, anti-ultraviolet damage, and anti-tumor properties. The manufacturing method of the astaxanthin nanovesicle carrier is to mix melanoma cells and astaxanthin in an appropriate ratio and then remove the genetic material and inflammatory factors of cancer cells by extrusion. Results: In terms of results, after the co-culture of astaxanthin nanovesicles and melanoma cancer cells, it was confirmed that the ability of astaxanthin nanovesicles to inhibit the growth and metastasis of melanoma cancer cells was significantly better than the same amount of astaxanthin alone, and it had no effect on normal Human cells are also effective. There was no apparent harm on normal cells, indicating the ability of the vesicles to be selectively transported. Conclusion: Our findings illustrated the potential of astaxanthin nanovesicles as an anticancer drug.


Assuntos
Antineoplásicos , Melanoma , Nanopartículas , Humanos , Melanoma/tratamento farmacológico , Antineoplásicos/farmacologia , Membrana Celular , Xantofilas
7.
Front Cell Infect Microbiol ; 14: 1346565, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469346

RESUMO

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.


Assuntos
Anti-Infecciosos , Infecções por Bactérias Gram-Negativas , Stenotrophomonas maltophilia , Humanos , Stenotrophomonas maltophilia/metabolismo , Bactérias Gram-Negativas , Biofilmes , Membrana Celular , Anti-Infecciosos/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia
8.
Biotechnol J ; 19(3): e2300654, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38472089

RESUMO

Vigorous ex vivo expansion of NK-92 cells is a pivotal step for clinical adoptive immunotherapy. Interleukin-2 (IL-2) is identified as a key cytokine for NK-92 cells, and it can stimulate cell proliferation after binding to the IL-2 receptor (IL-2R). In this work, the differences in IL-2 consumption and IL-2R expression were investigated between the two culture modes. The results showed that suspension culture favored ex vivo expansion of NK-92 cells compared with static culture. The specific consumption rate of IL-2 in suspension culture was significantly higher than that in static culture. It was further found that the mRNA levels of the two IL-2R subunits remained unchanged in suspension culture, but the proportion of NK-92 cells expressing IL-2Rß was increased, and the fluorescence intensity of IL-2Rß was remarkably enhanced. Meanwhile, the proportion of cells expressing IL-2R receptor complex also increased significantly. Correspondingly, the phosphorylation of STAT5, a pivotal protein in the downstream signaling pathway of IL-2, was up-regulated. Notably, the expression level and colocalization coefficient of related endosomes during IL-2/IL-2R complex endocytosis were markedly elevated, suggesting the enhancement of IL-2 endocytosis. Taken together, these results implied that more IL-2 was needed to support cell growth in suspension culture. Therefore, the culture process was optimized from the perspective of cytokine utilization to further improve the NK-92 cell's expansion ability and function. This study provides valuable insight into the efficient ex vivo expansion of NK-92 cells.


Assuntos
Interleucina-2 , Células Matadoras Naturais , Interleucina-2/metabolismo , Células Matadoras Naturais/metabolismo , Receptores de Interleucina-2/metabolismo , Citocinas/metabolismo , Membrana Celular
9.
Cells ; 13(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38474329

RESUMO

Wnt signaling is a highly conserved metazoan pathway that plays a crucial role in cell fate determination and morphogenesis during development. Wnt ligands can induce disparate cellular responses. The exact mechanism behind these different outcomes is not fully understood but may be due to interactions with different receptors on the cell membrane. PTK7/Otk is a transmembrane receptor that is implicated in various developmental and physiological processes including cell polarity, cell migration, and invasion. Here, we examine two roles of Otk-1 and Otk-2 in patterning and neurogenesis. We find that Otk-1 is a positive regulator of signaling and Otk-2 functions as its inhibitor. We propose that PTK7/Otk functions in signaling, cell migration, and polarity contributing to the diversity of cellular responses seen in Wnt-mediated processes.


Assuntos
Padronização Corporal , Neurogênese , Receptores Proteína Tirosina Quinases , Via de Sinalização Wnt , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Via de Sinalização Wnt/fisiologia
10.
Methods Mol Biol ; 2778: 43-52, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478270

RESUMO

Numerous bioinformatics tools allow predicting the localization of membrane proteins in the outer or inner membrane of Escherichia coli with high precision. Nevertheless, it might be desirable to experimentally verify such predictions or to assay the correct localization of recombinant or mutated variants of membrane proteins. Here we describe two methods (preferential detergent solubilization and sucrose-gradient fractionation) that allow to fractionate Gram-negative bacterial membranes and subsequently to enrich inner or outer membrane proteins.


Assuntos
Escherichia coli , Proteínas de Membrana , Membrana Celular , Escherichia coli/genética , Bactérias Gram-Negativas , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias , Fracionamento Celular/métodos
11.
Methods Mol Biol ; 2778: 159-183, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478278

RESUMO

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential ß-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Dobramento de Proteína
12.
Methods Mol Biol ; 2778: 133-145, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478276

RESUMO

Membrane-embedded ß-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. ß-barrels are highly structured domains formed by a series of antiparallel ß-strands. Each ß-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane ß-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane ß-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane ß-barrels.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Eletroforese em Gel de Poliacrilamida Nativa , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo
13.
Methods Mol Biol ; 2778: 367-381, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478289

RESUMO

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of ß-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo
14.
Sci Adv ; 10(11): eadj1512, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38478607

RESUMO

Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons.


Assuntos
Retículo Endoplasmático , Septinas , Septinas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto/metabolismo
15.
J Cell Biol ; 223(5)2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38470362

RESUMO

The eukaryotic p24 family, consisting of α-, ß-, γ- and δ-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion and that their localizations between ER exit site (ERES) and Golgi are interdependent in an α→ßδ→γ sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Proteínas de Drosophila , Retículo Endoplasmático , Complexo de Golgi , Proteínas de Membrana Transportadoras , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Membrana Celular , Drosophila melanogaster , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Domínios de Homologia de src , Proteínas de Membrana Transportadoras/metabolismo
16.
Sci Rep ; 14(1): 6031, 2024 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472404

RESUMO

The dysfunction of ion channels is a causative factor in a variety of neurological diseases, thereby defining the implicated channels as key drug targets. The detection of functional changes in multiple specific ionic currents currently presents a challenge, particularly when the neurological causes are either a priori unknown, or are unexpected. Traditional patch clamp electrophysiology is a powerful tool in this regard but is low throughput. Here, we introduce a single-shot method for detecting alterations amongst a range of ion channel types from subtle changes in membrane voltage in response to a short chaotically driven current clamp protocol. We used data assimilation to estimate the parameters of individual ion channels and from these we reconstructed ionic currents which exhibit significantly lower error than the parameter estimates. Such reconstructed currents thereby become sensitive predictors of functional alterations in biological ion channels. The technique correctly predicted which ionic current was altered, and by approximately how much, following pharmacological blockade of BK, SK, A-type K+ and HCN channels in hippocampal CA1 neurons. We anticipate this assay technique could aid in the detection of functional changes in specific ionic currents during drug screening, as well as in research targeting ion channel dysfunction.


Assuntos
Canais Iônicos , Neurônios , Eletrofisiologia , Canais Iônicos/metabolismo , Neurônios/metabolismo , Membrana Celular/metabolismo , Transporte de Íons
17.
Cell Mol Life Sci ; 81(1): 129, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472514

RESUMO

Recent work putatively linked a rare genetic variant of the chaperone Resistant to Inhibitors of acetylcholinesterase (RIC3) (NM_024557.4:c.262G > A, NP_078833.3:p.G88R) to a unique ability to speak backwards, a language skill that is associated with exceptional working memory capacity. RIC3 is important for the folding, maturation, and functional expression of α7 nicotinic acetylcholine receptors (nAChR). We compared and contrasted the effects of RIC3G88R on assembly, cell surface expression, and function of human α7 receptors using fluorescent protein tagged α7 nAChR and Förster resonance energy transfer (FRET) microscopy imaging in combination with functional assays and 125I-α-bungarotoxin binding. As expected, the wild-type RIC3 protein was found to increase both cell surface and functional expression of α7 receptors. In contrast, the variant form of RIC3 decreased both. FRET analysis showed that RICG88R increased the interactions between RIC3 and α7 protein in the endoplasmic reticulum. These results provide interesting and novel data to show that a RIC3 variant alters the interaction of RIC3 and α7, which translates to decreased cell surface and functional expression of α7 nAChR.


Assuntos
Receptores Nicotínicos , Humanos , Acetilcolinesterase/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Nicotínicos/genética , Fala
18.
Int J Mol Sci ; 25(5)2024 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-38473831

RESUMO

A critical determinant of infectivity and virulence of the most infectious and or lethal variants of concern (VOCs): Wild Type, Delta and Omicron is related to the binding interactions between the receptor-binding domain of the spike and its host receptor, the initial step in cell infection. It is of the utmost importance to understand how mutations of a viral strain, especially those that are in the viral spike, affect the resulting infectivity of the emerging VOC, knowledge that could help us understand the variant virulence and inform the therapies applied or the vaccines developed. For this sake, we have applied a battery of computational protocols of increasing complexity to the calculation of the spike binding affinity for three variants of concern to the ACE2 cell receptor. The results clearly illustrate that the attachment of the spikes of the Delta and Omicron variants to the receptor originates through different molecular interaction mechanisms. All our protocols unanimously predict that the Delta variant has the highest receptor-binding affinity, while the Omicron variant displays a substantial variability in the binding affinity of the spike that relates to the structural plasticity of the Omicron spike-receptor complex. We suggest that the latter result could explain (at least in part) the variability of the in vitro binding results for this VOC and has led us to suggest a reason for the lower virulence of the Omicron variant as compared to earlier strains. Several hypotheses have been developed around this subject.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Receptores de Superfície Celular , Membrana Celular , Mutação
19.
Int J Mol Sci ; 25(5)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38474126

RESUMO

CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described CD177 c.1291G>A variant on CD177 expression. Recombinant CD177 c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The CD177 c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that CD177 c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of CD177 c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.


Assuntos
Isoantígenos , Receptores de Superfície Celular , Humanos , Receptores de Superfície Celular/metabolismo , Proteínas Ligadas por GPI/metabolismo , Alelos , Membrana Celular/metabolismo , Mieloblastina/genética , Fenótipo , Isoantígenos/genética , Neutrófilos/metabolismo
20.
Int J Mol Sci ; 25(5)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38474151

RESUMO

Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs.


Assuntos
Vesículas Extracelulares , Bactérias Gram-Positivas , Bactérias , Membranas , Membrana Celular , Bicamadas Lipídicas/metabolismo , Vesículas Extracelulares/metabolismo
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