Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 92.024
Filtrar
1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
2.
J Phys Chem Lett ; 12(34): 8164-8169, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34410720

RESUMO

GPCR oligomerization plays a critical role in cellular signaling, yet the stoichiometry of the interactions between oligomers and binding ligands in living cells remains a longstanding challenge. Here, by developing a dual-color simultaneous tracking system based on a total internal reflection fluorescence microscope (TIRFM), the CCR5-CCL5 interactions are visualized and quantitatively assessed in real time. Results show that each oligomeric state of CCR5 could bind with CCL5 but with different binding affinities; CCR5 dimers have a 3.5-fold higher binding affinity than the monomers. The dimerization may cause an asymmetric conformational change which makes the first binding pocket have a 3.5-fold higher binding affinity and the second have only a half compared with the monomeric CCR5. This study is the first example to directly scrutinize the CCR5-CCL5 interactions at the single-molecule level on living cell membranes and will offer great potential for the interaction stoichiometry study of diverse surface proteins.


Assuntos
Membrana Celular/metabolismo , Multimerização Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Linhagem Celular , Cor , Ligantes , Ligação Proteica , Estrutura Quaternária de Proteína
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445285

RESUMO

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.


Assuntos
Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Peptídeos/metabolismo , Receptores de AMPA/metabolismo , Análise de Sequência de Proteína , Acetilglucosamina/genética , Animais , Membrana Celular/genética , Glicosilação , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Mapeamento de Peptídeos , Peptídeos/genética , Receptores de AMPA/genética
4.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445298

RESUMO

Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.


Assuntos
Membrana Celular/metabolismo , Receptores da Família Eph/química , Receptores da Família Eph/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/química , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos/fisiologia
5.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445358

RESUMO

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.


Assuntos
Escherichia coli/genética , Engenharia de Proteínas/métodos , Receptores Dopaminérgicos/genética , Calibragem , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Biblioteca Gênica , Humanos , Mutação , Organismos Geneticamente Modificados , Engenharia de Proteínas/normas , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Bacteriana , Transgenes
6.
Nat Commun ; 12(1): 4983, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404783

RESUMO

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite's apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.


Assuntos
Organelas/metabolismo , Organelas/ultraestrutura , Parasitos/metabolismo , Parasitos/ultraestrutura , Proteínas de Protozoários/química , Animais , Evolução Biológica , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Criptosporidiose , Cryptosporidium , Cryptosporidium parvum/citologia , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/metabolismo , Interações Hospedeiro-Parasita , Microtúbulos/ultraestrutura , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose
7.
Nat Commun ; 12(1): 4990, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404808

RESUMO

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Cálcio/metabolismo , Citoplasma , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Membranas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo
8.
Nat Commun ; 12(1): 4838, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376698

RESUMO

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 3-Quinases/metabolismo , Pinocitose/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura , Morfolinas/farmacologia , Fosfatidilinositóis/metabolismo , Pinocitose/efeitos dos fármacos , Células RAW 264.7
9.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445768

RESUMO

The two-pore domain K2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K+ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.


Assuntos
Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Fenômenos Físicos
10.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360941

RESUMO

Phospholipids (PLs) are amphiphilic molecules that were essential for life to become cellular. PLs have not only a key role in compartmentation as they are the main components of membrane, but they are also involved in cell signaling, cell metabolism, and even cell pathophysiology. Considered for a long time to simply be structural elements of membranes, phospholipids are increasingly being viewed as sensors of their environment and regulators of many metabolic processes. After presenting their main characteristics, we expose the increasing methods of PL detection and identification that help to understand their key role in life processes. Interest and importance of PL homeostasis is growing as pathogenic variants in genes involved in PL biosynthesis and/or remodeling are linked to human diseases. We here review diseases that involve deregulation of PL homeostasis and present a predominantly muscular phenotype.


Assuntos
Músculo Estriado/metabolismo , Fosfolipídeos/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Músculo Estriado/fisiologia , Fosfolipídeos/química
11.
J Chem Theory Comput ; 17(8): 5342-5357, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339605

RESUMO

The realism and accuracy of lipid bilayer simulations through molecular dynamics (MD) are heavily dependent on the lipid composition. While the field is pushing toward implementing more heterogeneous and realistic membrane compositions, a lack of high-resolution lipidomic data prevents some membrane protein systems from being modeled with the highest level of realism. Given the additional diversity of real-world cellular membranes and protein-lipid interactions, it is still not fully understood how altering membrane complexity affects modeled membrane protein functions or if it matters over long-timescale simulations. This is especially true for organisms whose membrane environments have little to no computational study, such as the plant plasma membrane. Tackling these issues in tandem, a generalized, realistic, and asymmetric plant plasma membrane with more than 10 different lipid species is constructed herein. Classical MD simulations of pure membrane constructs were performed to evaluate how altering the compositional complexity of the membrane impacted the plant membrane properties. The apo form of a plant sugar transporter, OsSWEET2b, was inserted into membrane models where lipid diversity was calculated in either a size-dependent or size-independent manner. An adaptive sampling simulation regime validated by Markov-state models was performed to capture the gating dynamics of OsSWEET2b in each of these membrane constructs. In comparison to previous OsSWEET2b simulations performed in a pure POPC bilayer, we confirm that simulations performed within a native-like membrane composition alter the stabilization of apo OsSWEET2b conformational states by ∼1 kcal/mol. The free-energy barriers of intermediate conformational states decrease when realistic membrane complexity is simplified, albeit roughly within sampling error, suggesting that protein-specific responses to membranes differ due to altered packing caused by compositional fluctuations. This work serves as a case study where a more realistic bilayer composition makes unbiased conformational sampling easier to achieve than with simplified bilayers.


Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
12.
Int J Mol Sci ; 22(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34445418

RESUMO

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.


Assuntos
Perfilação da Expressão Gênica/métodos , Nephropidae/genética , Neuropeptídeos/farmacologia , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sistema Cardiovascular/metabolismo , Membrana Celular/metabolismo , Clonagem Molecular , Mineração de Dados , Bases de Dados Genéticas , Regulação da Expressão Gênica/efeitos dos fármacos , Miocárdio/metabolismo , Nephropidae/efeitos dos fármacos , Nephropidae/metabolismo , Análise de Sequência de RNA , Células Sf9 , Distribuição Tecidual
13.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443456

RESUMO

Guanidinyl tryptophan derivatives TGN1, TGN2, TGN3, and TGN4 were synthesized, and these compounds were shown to possess in vitro inhibitory activity for amyloid aggregation in a previous study. Nevertheless, the influence of the TGN series of compounds on the binding and permeation behaviors of an Aß monomer to the cell membranes was not elucidated. In this study, we investigated the effect of compounds in the TGN series on the behavior of an Aß monomer regarding its toxicity toward the bilayer lipid membrane using molecular dynamics (MD) simulation. MD simulations suggest that TGN4 is a potential agent that can interfere with the movement of the Aß monomer into the membrane. The MM-GBSA result demonstrated that TGN4 exhibits the highest affinity to the Aß1-42 monomer but has the lowest affinity to the bilayer. Moreover, TGN4 also contributes to a decrease in the binding affinity between the Aß1-42 monomer and the POPC membrane. Regarding the results of the binding mode and conformational analyses, a high number of amino-acid residues were shown to provide the binding interactions between TGN4 and the Aß1-42 monomer. TGN4 also reduces the conformational transition of the Aß1-42 monomer by means of interacting with the monomer. The present study presents molecular-level insights into how the TGN series of compounds affect the membrane adsorption and the conformational transition of the Aß1-42 monomer, which could be valuable for the further development of new anti-Alzheimer agents.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Membrana Celular/metabolismo , Guanidina/uso terapêutico , Triptofano/uso terapêutico , Adesividade , Adsorção , Guanidina/química , Humanos , Ligantes , Bicamadas Lipídicas/química , Lipídeos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Conformação Proteica , Estrutura Secundária de Proteína , Triptofano/química , Água/química
14.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443459

RESUMO

Antioxidants remain interesting molecules of choice for suppression of the toxic effects of free radicals in foods and human systems. The current practice involves the use of mainly synthetic molecules as potent antioxidant agents. However, due to the potential negative impact on human health, there is an intensive effort within the research community to develop natural alternatives with similar antioxidant efficacy but without the negative side effects of synthetic molecules. Still, the successful development of new molecules depends on the use of reliable chemical or cell culture assays to screen antioxidant properties. Chemical antioxidant assays include the determination of scavenging ability against free radicals such as DPPH, superoxide anion radicals, hydroxyl radicals, hydrogen peroxide, and nitric oxide. Other antioxidant tests include the ability of compounds to bind and sequester prooxidant metal cations, reduce ferric iron, and attenuate the rate of lipid oxidation. Ex vivo tests utilize cell cultures to confirm entry of the molecules into cells and the ability to quench synthetic intracellular free radicals or to stimulate the increased biosynthesis of endogenous antioxidants. In order to assist researchers in their choice of antioxidant evaluation methods, this review presents background scientific information on some of the most commonly used antioxidant assays with a comparative discussion of the relevance of published literature data to food science and human nutrition applications.


Assuntos
Antioxidantes/metabolismo , Bioensaio/métodos , Células/metabolismo , Antioxidantes/química , Membrana Celular/metabolismo , Humanos , Peroxidação de Lipídeos , Espécies Reativas de Oxigênio/metabolismo
15.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360932

RESUMO

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Antígeno CD24/metabolismo , Membrana Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos
16.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445487

RESUMO

Mechanosensitive ion channels are widely expressed in the cardiovascular system. They translate mechanical forces including shear stress and stretch into biological signals. The most prominent biological signal through which the cardiovascular physiological activity is initiated or maintained are intracellular calcium ions (Ca2+). Growing evidence show that the Ca2+ entry mediated by mechanosensitive ion channels is also precisely regulated by a variety of key proteins which are distributed in the cell membrane or endoplasmic reticulum. Recent studies have revealed that mechanosensitive ion channels can even physically interact with Ca2+ regulatory proteins and these interactions have wide implications for physiology and pathophysiology. Therefore, this paper reviews the cross-talk between mechanosensitive ion channels and some key Ca2+ regulatory proteins in the maintenance of calcium homeostasis and its relevance to cardiovascular health and disease.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/metabolismo , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Homeostase , Humanos
17.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361083

RESUMO

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl- ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.


Assuntos
Proteínas de Transporte de Ânions/química , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Transportadores de Sulfato/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions/metabolismo , Sítios de Ligação , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência , Transportadores de Sulfato/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
18.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361115

RESUMO

DivIVA is a protein initially identified as a spatial regulator of cell division in the model organism Bacillus subtilis, but its homologues are present in many other Gram-positive bacteria, including Clostridia species. Besides its role as topological regulator of the Min system during bacterial cell division, DivIVA is involved in chromosome segregation during sporulation, genetic competence, and cell wall synthesis. DivIVA localizes to regions of high membrane curvature, such as the cell poles and cell division site, where it recruits distinct binding partners. Previously, it was suggested that negative curvature sensing is the main mechanism by which DivIVA binds to these specific regions. Here, we show that Clostridioides difficile DivIVA binds preferably to membranes containing negatively charged phospholipids, especially cardiolipin. Strikingly, we observed that upon binding, DivIVA modifies the lipid distribution and induces changes to lipid bilayers containing cardiolipin. Our observations indicate that DivIVA might play a more complex and so far unknown active role during the formation of the cell division septal membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Cardiolipinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Clostridioides difficile/metabolismo , Lipídeos de Membrana/metabolismo , Clostridioides difficile/crescimento & desenvolvimento , Transporte Proteico
19.
Nat Commun ; 12(1): 4707, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349110

RESUMO

Salmonella utilizes translocated virulence proteins (termed effectors) to promote host cell invasion. The effector SopD contributes to invasion by promoting scission of the plasma membrane, generating Salmonella-containing vacuoles. SopD is expressed in all Salmonella lineages and plays important roles in animal models of infection, but its host cell targets are unknown. Here we show that SopD can bind to and inhibit the small GTPase Rab10, through a C-terminal GTPase activating protein (GAP) domain. During infection, Rab10 and its effectors MICAL-L1 and EHBP1 are recruited to invasion sites. By inhibiting Rab10, SopD promotes removal of Rab10 and recruitment of Dynamin-2 to drive scission of the plasma membrane. Together, our study uncovers an important role for Rab10 in regulating plasma membrane scission and identifies the mechanism used by a bacterial pathogen to manipulate this function during infection.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Salmonella typhimurium/patogenicidade , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas de Bactérias/genética , Dinamina II , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Salmonella typhimurium/metabolismo , Vacúolos/metabolismo , Vacúolos/microbiologia , Virulência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
20.
Nat Commun ; 12(1): 4697, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349123

RESUMO

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.


Assuntos
Antígenos CD13/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Epitélio/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD13/química , Antígenos CD13/genética , Células CACO-2 , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Proteínas rab de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...