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1.
Nat Commun ; 12(1): 5106, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429434

RESUMO

The cytoplasmic domain of PD-L1 (PD-L1-CD) regulates PD-L1 degradation and stability through various mechanism, making it an attractive target for blocking PD-L1-related cancer signaling. Here, by using NMR and biochemical techniques we find that the membrane association of PD-L1-CD is mediated by electrostatic interactions between acidic phospholipids and basic residues in the N-terminal region. The absence of the acidic phospholipids and replacement of the basic residues with acidic residues abolish the membrane association. Moreover, the basic-to-acidic mutations also decrease the cellular abundance of PD-L1, implicating that the electrostatic interaction with the plasma membrane mediates the cellular levels of PD-L1. Interestingly, distinct from its reported function as an activator of AMPK in tumor cells, the type 2 diabetes drug metformin enhances the membrane dissociation of PD-L1-CD by disrupting the electrostatic interaction, thereby decreasing the cellular abundance of PD-L1. Collectively, our study reveals an unusual regulatory mechanism that controls the PD-L1 level in tumor cells, suggesting an alternative strategy to improve the efficacy of PD-L1-related immunotherapies.


Assuntos
Antígeno B7-H1/metabolismo , Membranas/metabolismo , Eletricidade Estática , Antígeno B7-H1/química , Antígeno B7-H1/genética , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2 , Células HEK293 , Humanos , Imunoterapia , Metformina , Mutação
2.
Nat Commun ; 12(1): 4972, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404795

RESUMO

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.


Assuntos
Fusão de Membrana/fisiologia , Membranas/metabolismo , Polímeros/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Animais , Microscopia Crioeletrônica , Lipossomos/metabolismo , Proteínas do Tecido Nervoso , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteína 2 Associada à Membrana da Vesícula
3.
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
4.
Methods Mol Biol ; 2315: 43-57, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302669

RESUMO

Protein engineering can yield new molecular tools for nanotechnology and therapeutic applications through modulating physiochemical and biological properties. Engineering membrane proteins is especially attractive because they perform key cellular processes including transport, nutrient uptake, removal of toxins, respiration, motility, and signaling. In this chapter, we describe two protocols for membrane protein engineering with the Rosetta software: (1) ΔΔG calculations for single point mutations and (2) sequence optimization in different membrane lipid compositions. These modular protocols are easily adaptable for more complex problems and serve as a foundation for efficient membrane protein engineering calculations.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo , Engenharia de Proteínas/métodos , Transporte Biológico/fisiologia , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Software
5.
Methods Mol Biol ; 2315: 243-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302680

RESUMO

In silico simulations of biological systems are of the significant importance to obtain insights on specific processes that experimental protocols have difficulty to elucidate. More particularly, and to ensure that a given molecule is able to reach its cellular target, the development of computational methods able to quickly estimate the cellular permeabilities for small molecules can become an important tool in the early stages of drug development. Herein, a computational protocol for predicting permeability coefficients, concerning both membranes and proteins, is presented and discussed.


Assuntos
Biologia Computacional/métodos , Bicamadas Lipídicas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Membranas/metabolismo , Simulação de Dinâmica Molecular , Permeabilidade , Proteínas/metabolismo
6.
Methods Mol Biol ; 2315: 263-271, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302681

RESUMO

Pan-assay interference compounds (PAINS) are promiscuous molecules with multiple behaviors that interfere with assay readouts. Membrane PAINS are a subset of these compounds that influence the function of membrane proteins by nonspecifically perturbing the lipid membranes that surround them. Here, we describe a computational protocol to identify potential membrane PAINS molecules by calculating the effect that a given compound has on the bilayer deformation propensity.


Assuntos
Biologia Computacional/métodos , Descoberta de Drogas/métodos , Preparações Farmacêuticas/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismo
7.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069905

RESUMO

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.


Assuntos
Orthomyxoviridae/metabolismo , Proteínas Virais de Fusão/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Bicamadas Lipídicas/química , Lipossomos/metabolismo , Fusão de Membrana , Membranas/metabolismo , Modelos Teóricos , Simulação de Dinâmica Molecular , Orthomyxoviridae/patogenicidade , Proteínas Virais de Fusão/química
8.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
9.
Cell Death Dis ; 12(4): 360, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824270

RESUMO

In the early 1990s, it has been described that LTα and LTß form LTα2ß and LTαß2 heterotrimers, which bind to TNFR1 and LTßR, respectively. Afterwards, the LTαß2-LTßR system has been intensively studied while the LTα2ß-TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα2ß-TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα2ß interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα2ß (memLTα2ß), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα2ß is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.


Assuntos
Linfotoxina-alfa/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/agonistas , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Membranas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/agonistas , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologia
10.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807215

RESUMO

The water environment determines the activity of biological processes. The role of such an environment interpreted in the form of an external field expressed by the 3D Gaussian distribution in the fuzzy oil drop model directs the folding process towards the generation of a centrally located hydrophobic core with the simultaneous exposure of polar residues on the surface. In addition to proteins soluble in the water environment, there is a significant group of membrane proteins that act as receptors or channels, including ion channels in particular. The change of the polar (water) environment into a highly hydrophobic (membrane) environment is quite radical, resulting in a different hydrophobicity distribution within the membrane protein. Modification of the notation of the force field expressing the presence of the hydrophobic environment has been proposed in this work. A modified fuzzy oil drop model with its adaptation to membrane proteins was used to interpret the structure of membrane proteins-mechanosensitive channel. The modified model was also used to describe the so-called negative cases-i.e., for water-soluble proteins with a clear distribution consistent with the fuzzy oil drop model.


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/química , Água/química , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Membranas/química , Membranas/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína
11.
Mol Biol Cell ; 32(7): 521-537, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33566711

RESUMO

Before their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the endoplasmic reticulum (ER) and inner-nuclear membrane (INM) must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the INM, which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ER-associated degradation (ERAD)-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel that can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.


Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Membrana Nuclear/metabolismo , Proteostase/fisiologia , Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
12.
Phys Rev E ; 103(1-1): 013108, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33601537

RESUMO

Mucin polymers in the tear film protect the corneal surface from pathogens and modulate the tear-film flow characteristics. Recent studies have suggested a relationship between the loss of membrane-associated mucins and premature rupture of the tear film in various eye diseases. This work aims to elucidate the hydrodynamic mechanisms by which loss of membrane-associated mucins causes premature tear-film rupture. We model the bulk of the tear film as a Newtonian fluid in a two-dimensional periodic domain, and the lipid layer at the air-tear interface as insoluble surfactants. Gradual loss of membrane-associated mucins produces growing areas of exposed cornea in direct contact with the tear fluid. We represent the hydrodynamic consequences of this morphological change through two mechanisms: an increased van der Waals attraction due to loss of wettability on the exposed area, and a change of boundary condition from an effective negative slip on the mucin-covered areas to the no-slip condition on exposed cornea. Finite-element computations, with an arbitrary Lagrangian-Eulerian scheme to handle the moving interface, demonstrate a strong effect of the elevated van der Waals attraction on precipitating tear-film breakup. The change in boundary condition on the cornea has a relatively minor role. Using realistic parameters, our heterogeneous mucin model is able to predict quantitatively the shortening of tear-film breakup time observed in diseased eyes.


Assuntos
Membranas/metabolismo , Modelos Biológicos , Mucinas/metabolismo
13.
Theranostics ; 11(7): 3183-3195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537081

RESUMO

Exosomes are cell-derived nanovesicles that are involved in the intercellular transportation of materials. Therapeutics, such as small molecules or nucleic acid drugs, can be incorporated into exosomes and then delivered to specific types of cells or tissues to realize targeted drug delivery. Targeted delivery increases the local concentration of therapeutics and minimizes side effects. Here, we present a detailed review of exosomes engineering through genetic and chemical methods for targeted drug delivery. Although still in its infancy, exosome-mediated drug delivery boasts low toxicity, low immunogenicity, and high engineerability, and holds promise for cell-free therapies for a wide range of diseases.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Exossomos/metabolismo , Engenharia de Proteínas/métodos , Animais , Exossomos/química , Exossomos/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Membranas/química , Membranas/metabolismo
14.
Methods Mol Biol ; 2251: 91-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481233

RESUMO

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLCδ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.


Assuntos
Membrana Celular/química , Fosfatidilinositóis/análise , Imagem Individual de Molécula/métodos , Animais , Técnicas de Cultura de Células/métodos , Membrana Celular/metabolismo , Humanos , Membranas/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Transporte Proteico/fisiologia , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/química , Fosfolipases Tipo C/metabolismo
15.
Methods Mol Biol ; 2251: 195-204, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481241

RESUMO

A large proportion of proteins are expected to interact with cellular membranes to carry out their physiological functions in processes such as membrane transport, morphogenesis, cytoskeletal organization, and signal transduction. The recruitment of proteins at the membrane-cytoplasm interface and their activities are precisely regulated by phosphoinositides, which are negatively charged phospholipids found on the cytoplasmic leaflet of cellular membranes and play critical roles in membrane homeostasis and cellular signaling. Thus, it is important to reveal which proteins interact with phosphoinositides and to elucidate the underlying mechanisms. Here, we present two standard in vitro methods, liposome co-sedimentation and co-flotation assays, to study lipid-protein interactions. Liposomes can mimic various biological membranes in these assays because their lipid compositions and concentrations can be varied. Thus, in addition to mechanisms of lipid-protein interactions, these methods provide information on the possible specificities of proteins toward certain lipids such as specific phosphoinositide species and can hence shed light on the roles of membrane interactions on the functions of membrane-associated proteins.


Assuntos
Lipossomos/análise , Fosfatidilinositóis/análise , Mapeamento de Interação de Proteínas/métodos , Membrana Celular/metabolismo , Humanos , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas/química , Transdução de Sinais/fisiologia
16.
Sci Rep ; 11(1): 906, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33441861

RESUMO

GMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.


Assuntos
Antígenos O/imunologia , Shigella sonnei/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Disenteria Bacilar/prevenção & controle , Disenteria Bacilar/terapia , Exossomos/imunologia , Feminino , Imunoglobulina G/metabolismo , Membranas/metabolismo , Camundongos , Antígenos O/química , Antígenos O/metabolismo , Proteômica/métodos , Shigella sonnei/patogenicidade , Vacinação/métodos , Vacinas/imunologia
17.
Methods Mol Biol ; 2251: 143-156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481237

RESUMO

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.


Assuntos
Membranas/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Fosfolipídeos/análise , Animais , Humanos , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Microfluídica/métodos , Fosfatidilinositóis/metabolismo , Fosfolipídeos/química , Ligação Proteica/fisiologia , Proteínas/metabolismo
18.
Cell Microbiol ; 23(1): e13267, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32975360

RESUMO

Rab small GTPases regulate membrane traffic between distinct cellular compartments of all eukaryotes in a tempo-spatially specific fashion. Rab small GTPases are also involved in the regulation of cytoskeleton and signalling. Membrane traffic and cytoskeletal regulation play pivotal role in the pathogenesis of Entamoeba histolytica, which is a protozoan parasite responsible for human amebiasis. E. histolytica is unique in that its genome encodes over 100 Rab proteins, containing multiple isotypes of conserved members (e.g., Rab7) and Entamoeba-specific subgroups (e.g., RabA, B, and X). Among them, E. histolytica Rab7 is the most diversified group consisting of nine isotypes. While it was previously demonstrated that EhRab7A and EhRab7B are involved in lysosome and phagosome biogenesis, the individual roles of other Rab7 members and their coordination remain elusive. In this study, we characterised the third member of Rab7, Rab7D, to better understand the significance of the multiplicity of Rab7 isotypes in E. histolytica. Overexpression of EhRab7D caused reduction in phagocytosis of erythrocytes, trogocytosis (meaning nibbling or chewing of a portion) of live mammalian cells, and phagosome acidification and maturation. Conversely, transcriptional gene silencing of EhRab7D gene caused opposite phenotypes in phago/trogocytosis and phagosome maturation. Furthermore, EhRab7D gene silencing caused reduction in the attachment to and the motility on the collagen-coated surface. Image analysis showed that EhRab7D was occasionally associated with lysosomes and prephagosomal vacuoles, but not with mature phagosomes and trogosomes. Finally, in silico prediction of structural organisation of EhRab7 isotypes identified unique amino acid changes on the effector binding surface of EhRab7D. Taken together, our data suggest that EhRab7D plays coordinated counteracting roles: a inhibitory role in phago/trogocytosis and lyso/phago/trogosome biogenesis, and an stimulatory role in adherence and motility, presumably via interaction with unique effectors. Finally, we propose the model in which three EhRab7 isotypes are sequentially involved in phago/trogocytosis.


Assuntos
Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fagocitose , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Citoesqueleto/metabolismo , Entamoeba histolytica/patogenicidade , Entamebíase/parasitologia , Inativação Gênica , Humanos , Lisossomos/metabolismo , Membranas/metabolismo , Fagossomos/metabolismo , Transcriptoma , Vacúolos/metabolismo , Virulência , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
19.
Clin Podiatr Med Surg ; 38(1): 99-110, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33220747

RESUMO

The induced membrane technique is a simple, effective, and reproducible treatment method for segmental bone defects. It is a 2-stage approach that requires eventual autologous bone graft to manage the deficit. The first stage requires debridement of all nonviable tissue while preserving a healthy soft tissue envelope. A polymethylmethacrylate is implanted between the osseous segments to maintain length. The osseous defect can be stabilized internally or externally. During the second stage, a vascularized induced membrane is formed and produces multiple growth factors. The induced membrane technique is a valuable option for limb salvage in cases of segmental bone defects.


Assuntos
Reação a Corpo Estranho , Fraturas Ósseas/cirurgia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Membranas/crescimento & desenvolvimento , Membranas/metabolismo , Lesões dos Tecidos Moles/cirurgia , Autoenxertos , Regeneração Óssea , Osso Esponjoso/transplante , Desbridamento , Humanos , Salvamento de Membro/métodos
20.
Int J Mol Sci ; 22(1)2020 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-33375246

RESUMO

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via 1H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.


Assuntos
Glicina/metabolismo , Glicilglicina/metabolismo , Membranas/metabolismo , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Água/metabolismo , Glicina/química , Glicilglicina/química , Membranas/química , Micelas , Modelos Teóricos , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Água/química
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