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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34000034

RESUMO

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.


Assuntos
Fosfatidilserinas , Saccharomyces cerevisiae , Membrana Celular , Microdomínios da Membrana , Fosfolipídeos , Saccharomyces cerevisiae/genética , Esfingolipídeos
2.
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
3.
Nature ; 597(7875): 220-224, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497391

RESUMO

A key aspect of living cells is their ability to harvest energy from the environment and use it to pump specific atomic and molecular species in and out of their system-typically against an unfavourable concentration gradient1. Active transport allows cells to store metabolic energy, extract waste and supply organelles with basic building blocks at the submicrometre scale. Unlike living cells, abiotic systems do not have the delicate biochemical machinery that can be specifically activated to precisely control biological matter2-5. Here we report the creation of microcapsules that can be brought out of equilibrium by simple global variables (illumination and pH), to capture, concentrate, store and deliver generic microscopic payloads. Borrowing no materials from biology, our design uses hollow colloids serving as spherical cell-membrane mimics, with a well-defined single micropore. Precisely tunable monodisperse capsules are the result of a synthetic self-inflation mechanism and can be produced in bulk quantities. Inside the hollow unit, a photoswitchable catalyst6 produces a chemical gradient that propagates to the exterior through the membrane's micropore and pumps target objects into the cell, acting as a phoretic tractor beam7. An entropic energy barrier8,9 brought about by the micropore's geometry retains the cargo even when the catalyst is switched off. Delivery is accomplished on demand by reversing the sign of the phoretic interaction. Our findings provide a blueprint for developing the next generation of smart materials, autonomous micromachinery and artificial cell-mimics.


Assuntos
Materiais Biomiméticos/metabolismo , Materiais Biomiméticos/efeitos da radiação , Biomimética , Membrana Celular/metabolismo , Coloides/metabolismo , Coloides/efeitos da radiação , Transporte Biológico Ativo/efeitos da radiação , Materiais Biomiméticos/química , Membrana Celular/efeitos da radiação , Coloides/química , Emulsões/química , Entropia , Concentração de Íons de Hidrogênio , Luz
4.
Molecules ; 26(17)2021 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-34500630

RESUMO

Necroptosis is a type of programmed cell death executed through the plasma membrane disruption by mixed lineage kinase domain-like protein (MLKL). Previous studies have revealed that an N-terminal four-helix bundle domain (NBD) of MLKL is the executioner domain for the membrane permeabilization, which is auto-inhibited by the first brace helix (H6). After necroptosis initiation, this inhibitory brace helix detaches and the NBD can integrate into the membrane, and hence leads to necroptotic cell death. However, how the NBD is released and induces membrane rupture is poorly understood. Here, we reconstituted MLKL2-154 into membrane mimetic bicelles and observed the structure disruption and membrane release of the first brace helix that is regulated by negatively charged phospholipids in a dose-dependent manner. Using molecular dynamics simulation we found that the brace region in an isolated, auto-inhibited MLKL2-154 becomes intrinsically disordered in solution after 7 ns dynamic motion. Further investigations demonstrated that a cluster of arginines in the C-terminus of MLKL2-154 is important for the molecular conformational switch. Functional mutagenesis showed that mutating these arginines to glutamates hindered the membrane disruption of full-length MLKL and thus inhibited the necroptotic cell death. These findings suggest that the brace helix also plays an active role in MLKL regulation, rather than an auto-inhibitory domain.


Assuntos
Membrana Celular/metabolismo , Necroptose/fisiologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Ácido Glutâmico/metabolismo , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/fisiologia
5.
Nat Commun ; 12(1): 5333, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504087

RESUMO

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.


Assuntos
COVID-19/metabolismo , Membrana Celular/metabolismo , Células Gigantes/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Proteômica , Células Vero , Montagem de Vírus/genética
6.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34526387

RESUMO

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from "close contacts" formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor's diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor "traps."


Assuntos
Comunicação Celular , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Humanos , Ligantes , Fosforilação , Linfócitos T/citologia
7.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34531300

RESUMO

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.


Assuntos
Moléculas de Adesão Celular/química , Adesão Celular , Síndrome de Down/patologia , Proteínas de Drosophila/química , Neurogênese , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Camundongos , Neuritos
8.
Toxicol Lett ; 351: 145-154, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34509610

RESUMO

Fe3O4 nanoparticles are widely used in the diagnosis and treatment of diseases due to their superparamagnetism, but their toxicity in vivo, which can result in apoptosis or autophagy, cannot be ignored. It has been reported that polydopamine (PDA) modification can reduce the toxicity of Fe3O4 and increase its biocompatibility. However, more research is warranted to further improve the modification method. We therefore developed a new method to coat Fe3O4@PDA nanoparticles with the mesenchymal stem cell membrane (MSCM) and evaluated the toxicity of the modified particles in the lungs of mice. We found that the MSCM modification significantly reduced lung injury induced by Fe3O4 particles in mice. Compared with Fe3O4@PDA nanoparticles, co-modification with MSCM and PDA significantly reduced autophagy and apoptosis in mouse lung tissue, and reduced activation of autophagy mediated by the AMPK-ULK1 pathway axis. Thus, co-modification with MSCM and PDA prevents Fe3O4-induced pulmonary toxicity in mice by inhibiting autophagy, apoptosis, and oxidative stress.


Assuntos
Adenilato Quinase/metabolismo , Membrana Celular/efeitos dos fármacos , Compostos Férricos/toxicidade , Indóis/farmacologia , Pneumopatias/induzido quimicamente , Células-Tronco Mesenquimais/efeitos dos fármacos , Polímeros/farmacologia , Adenilato Quinase/genética , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Membrana Celular/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos
9.
Cells ; 10(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34571822

RESUMO

As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention.


Assuntos
Esfingolipídeos/metabolismo , Viroses , Transporte Biológico , Membrana Celular/química , Ceramidas/metabolismo , Sistemas de Liberação de Medicamentos , HIV/crescimento & desenvolvimento , Interações entre Hospedeiro e Microrganismos , Membranas Intracelulares/química , SARS-CoV-2/crescimento & desenvolvimento , Vírion , Replicação Viral , Vírus/crescimento & desenvolvimento
10.
J Photochem Photobiol B ; 223: 112297, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34482154

RESUMO

Lipid oxidation is ubiquitous in cell life under oxygen and essential for photodynamic therapy (PDT) of carcinomas. However, the mechanisms underlying lipid oxidation in rather complex systems such as plasma membranes remain elusive. Herein, Langmuir monolayers were assembled with the lipid extract of glandular breast cancer (MCF7) cells and used to probe the molecular interactions allowing adsorption of the photosensitizer (PS) erythrosine B and subsequent photooxidation outcomes. Surface pressure (π) versus area (cm2/mL) isotherms of MCF7 lipid extract shifted to larger areas upon erythrosine incorporation, driven by secondary interactions that affected the orientation of the carbonyl groups and lipid chain organization. Light-irradiation increased the surface area of the MCF7 lipid extract monolayer containing erythrosine owing to the lipid hydroperoxidation, which may further undergo decomposition, resulting in the chain cleavage of phospholipids and membrane permeabilization. Incorporation of erythrosine by MCF7 cells induced slight toxic effects on in vitro assays, differently of the severe phototoxicity caused by light-irradiation, which significantly decreased cell viability by more than 75% at 2.5 × 10-6 mol/L of erythrosine incubated for 3 and 24 h, reaching nearly 90% at 48 h of incubation. The origin of the phototoxic effects is in the rupture of the plasma membrane shown by the frontal (FSC) and side (SSC) light scattering of flow cytometry. Consistent with hydroperoxide decomposition, membrane permeabilization was also confirmed by cleaved lipids detected in mass spectrometry and subsidizes the necrotic pathway of cell death.


Assuntos
Membrana Celular/efeitos dos fármacos , Eritrosina/farmacologia , Luz , Fármacos Fotossensibilizantes/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Elasticidade , Eritrosina/química , Feminino , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Lipídeos/análise , Lipídeos/química , Microscopia Confocal , Fármacos Fotossensibilizantes/química , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray
11.
Nat Commun ; 12(1): 5248, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504076

RESUMO

The HRAS, NRAS, and KRAS genes are collectively mutated in a fifth of all human cancers. These mutations render RAS GTP-bound and active, constitutively binding effector proteins to promote signaling conducive to tumorigenic growth. To further elucidate how RAS oncoproteins signal, we mined RAS interactomes for potential vulnerabilities. Here we identify EFR3A, an adapter protein for the phosphatidylinositol kinase PI4KA, to preferentially bind oncogenic KRAS. Disrupting EFR3A or PI4KA reduces phosphatidylinositol-4-phosphate, phosphatidylserine, and KRAS levels at the plasma membrane, as well as oncogenic signaling and tumorigenesis, phenotypes rescued by tethering PI4KA to the plasma membrane. Finally, we show that a selective PI4KA inhibitor augments the antineoplastic activity of the KRASG12C inhibitor sotorasib, suggesting a clinical path to exploit this pathway. In sum, we have discovered a distinct KRAS signaling axis with actionable therapeutic potential for the treatment of KRAS-mutant cancers.


Assuntos
Carcinogênese/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cães , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Fosfatos de Fosfatidilinositol/biossíntese , Fosfatidilserinas/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Commun Biol ; 4(1): 1086, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526645
13.
Phys Chem Chem Phys ; 23(36): 20627-20633, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34514475

RESUMO

Accumulation of protein-based (Aß) aggregates on cellular membranes with varying structural properties is commonly recognized as the key step in Alzheimer's pathogenesis. But experimental and computational challenges have made this biophysical characterization difficult. In particular, studies connecting biological membrane organization and Aß aggregation are limited. While experiments have suggested that an increased membrane curvature results in faster Aß peptide aggregation in the context of Alzheimer's disease, a mechanistic explanation for this relation is missing. In this work, we are leveraging molecular simulations with a physics-based coarse grained model to address and understand the relationships between curved cellular membranes and aggregation of a model template peptide Aß 16-22. In agreement with experimental results, our simulations also suggest a positive correlation between increased peptide aggregation and membrane curvature. More curved membranes have higher lipid packing defects that engage peptide hydrophobic groups and promote faster diffusion leading to peptide fibrillar structures. In addition, we curated the effects of peptide aggregation on the membrane's structure and organization. Interfacial peptide aggregation results in heterogeneous headgroup-peptide interactions and an induced crowding effect at the lipid headgroup region, leading to a more ordered headgroup region and disordered lipid-tails at the membrane core. This work presents a mechanistic and morphological overview of the relationships between the biomembrane local structure and organization, and Aß peptide aggregation.


Assuntos
Peptídeos beta-Amiloides/química , Membrana Celular , Humanos , Modelos Moleculares , Agregados Proteicos , Tensão Superficial
14.
Int J Med Sci ; 18(15): 3533-3543, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34522180

RESUMO

Importance: Despite the availability of a vaccine against the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), humans will have to live with this virus and the after-effects of the coronavirus disease 2019 (COVID-19) infection for a long time. Cholesterol plays an important role in the infection and prognosis of SARS-CoV-2, and the study of its mechanism is of great significance not only for the treatment of COVID-19 but also for research on generic antiviral drugs. Observations: Cholesterol promotes the development of atherosclerosis by activating NLR family pyrin domain containing 3 (NLRP3), and the resulting inflammatory environment indirectly contributes to COVID-19 infection and subsequent deterioration. In in vitro studies, membrane cholesterol increased the number of viral entry sites on the host cell membrane and the number of angiotensin-converting enzyme 2 (ACE2) receptors in the membrane fusion site. Previous studies have shown that the fusion protein of the virus interacts with cholesterol, and the spike protein of SARS-CoV-2 also requires cholesterol to enter the host cells. Cholesterol in blood interacts with the spike protein to promote the entry of spike cells, wherein the scavenger receptor class B type 1 (SR-B1) plays an important role. Because of the cardiovascular protective effects of lipid-lowering therapy and the additional anti-inflammatory effects of lipid-lowering drugs, it is currently recommended to continue lipid-lowering therapy for patients with COVID-19, but the safety of extremely low LDL-C is questionable. Conclusions and Relevance: Cholesterol can indirectly increase the susceptibility of patients to SARS-CoV-2 and increase the risk of death from COVID-19, which are mediated by NLRP3 and atherosclerotic plaques, respectively. Cholesterol present in the host cell membrane, virus, and blood may also directly participate in the virus cell entry process, but the specific mechanism still needs further study. Patients with COVID-19 are recommended to continue lipid-lowering therapy.


Assuntos
COVID-19/complicações , Hipercolesterolemia/complicações , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/uso terapêutico , Aterosclerose/fisiopatologia , COVID-19/diagnóstico , COVID-19/tratamento farmacológico , COVID-19/terapia , Membrana Celular/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Endocitose , Humanos , Hipercolesterolemia/diagnóstico , Hipercolesterolemia/terapia , Inflamação , Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/sangue , Prognóstico , SARS-CoV-2 , Receptores Depuradores Classe B/metabolismo
16.
Nat Commun ; 12(1): 5282, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489418

RESUMO

Homeostasis is one of the fundamental concepts in physiology. Despite remarkable progress in our molecular understanding of amino acid transport, metabolism and signaling, it remains unclear by what mechanisms cytosolic amino acid concentrations are maintained. We propose that amino acid transporters are the primary determinants of intracellular amino acid levels. We show that a cell's endowment with amino acid transporters can be deconvoluted experimentally and used this data to computationally simulate amino acid translocation across the plasma membrane. Transport simulation generates cytosolic amino acid concentrations that are close to those observed in vitro. Perturbations of the system are replicated in silico and can be applied to systems where only transcriptomic data are available. This work explains amino acid homeostasis at the systems-level, through a combination of secondary active transporters, functionally acting as loaders, harmonizers and controller transporters to generate a stable equilibrium of all amino acid concentrations.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Homeostase/genética , Modelos Estatísticos , Neuroglia/metabolismo , Células A549 , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/classificação , Sistemas de Transporte de Aminoácidos/genética , Animais , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Simulação por Computador , Expressão Gênica , Humanos , Cinética , Metabolômica/métodos , Neuroglia/citologia , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis
17.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544868

RESUMO

Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity of amyloid-ß peptides in Alzheimer's disease. To gain a detailed understanding of the mutual interference of amyloid-ß oligomers and the neuronal membrane, we carried out microseconds of all-atom molecular dynamics (MD) simulations on the dimerization of amyloid-ß (Aß)42 in the aqueous phase and in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The dimerization in solution is characterized by a random coil to ß-sheet transition that seems on pathway to amyloid aggregation, while the interactions with the neuronal membrane decrease the order of the Aß42 dimer by attenuating its propensity to form a ß-sheet structure. The main lipid interaction partners of Aß42 are the surface-exposed sugar groups of the gangliosides GM1. As the neurotoxic activity of amyloid oligomers increases with oligomer order, these results suggest that GM1 is neuroprotective against Aß-mediated toxicity.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/química , Membrana Celular/metabolismo , Gangliosídeo G(M1)/metabolismo , Neurônios/metabolismo , Multimerização Proteica , Humanos , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica
18.
Soft Matter ; 17(36): 8373-8386, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34550131

RESUMO

Membrane bending is an extensively studied problem from both modeling and experimental perspectives because of the wide implications of curvature generation in cell biology. Many of the curvature generating aspects in membranes can be attributed to interactions between proteins and membranes. These interactions include protein diffusion and formation of aggregates due to protein-protein interactions in the plane of the membrane. Recently, we developed a model that couples the in-plane flow of lipids and diffusion of proteins with the out-of-plane bending of the membrane. Building on this work, here, we focus on the role of explicit aggregation of proteins on the surface of the membrane in the presence of membrane bending and diffusion. We develop a comprehensive framework that includes lipid flow, membrane bending, the entropy of protein distribution, along with an explicit aggregation potential and derive the governing equations for the coupled system. We compare this framework to the Cahn-Hillard formalism to predict the regimes in which the proteins form patterns on the membrane. We demonstrate the utility of this model using numerical simulations to predict how aggregation and diffusion, when coupled with curvature generation, can alter the landscape of membrane-protein interactions.


Assuntos
Bicamadas Lipídicas , Proteínas , Membrana Celular , Difusão , Retroalimentação
19.
Biomed Res Int ; 2021: 6888494, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34540996

RESUMO

Aquaporins (AQPs) are proteins that selectively transport water across the cell membrane. Although AQPs play important roles in secretion in the lacrimal gland, the expression and localization of AQPs have not been clarified yet. In the current study, we investigated the expression pattern of AQP family members in the murine lacrimal gland during development. Lacrimal gland tissues were harvested from E13.5 and E17.5 murine embryos and from mice 8 weeks of age (adults). Corneal and conjunctival tissues from the latter served as controls. Total RNA was isolated and analyzed for the expression of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Expression of Aqp8 and Aqp9 mRNAs was detected in the adult lacrimal gland but not in the cornea, conjunctiva, or fetal lacrimal gland. AQP8 and AQP9 and α-SMA partially colocalized around the basal regions of the acinar unit. The levels of Aqp3 mRNAs and protein were much lower in the adult lacrimal gland but were readily detected in the adult cornea and conjunctiva. Our study suggests that AQP8 and AQP9 may serve as markers for adult murine lacrimal gland, ductal, and myoepithelial cells.


Assuntos
Aquaporinas/metabolismo , Aparelho Lacrimal/citologia , Fatores Etários , Animais , Aquaporinas/análise , Aquaporinas/fisiologia , Membrana Celular/metabolismo , Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/genética , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Transcriptoma/genética
20.
Nat Commun ; 12(1): 5426, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521824

RESUMO

Much hope in drug development comes from the discovery of positive allosteric modulators (PAM) that display target subtype selectivity and act by increasing agonist potency and efficacy. How such compounds can allosterically influence agonist action remains unclear. Metabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases, and for which PAMs acting in the transmembrane domain have been developed. Here, we explore the effect of a PAM on the structural dynamics of mGlu2 in optimized detergent micelles using single molecule FRET at submillisecond timescales. We show that glutamate only partially stabilizes the extracellular domains in the active state. Full activation is only observed in the presence of a PAM or the Gi protein. Our results provide important insights on the role of allosteric modulators in mGlu activation, by stabilizing the active state of a receptor that is otherwise rapidly oscillating between active and inactive states.


Assuntos
Ácido Glutâmico/farmacologia , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/química , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Aminoácidos/química , Aminoácidos/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Domínio Catalítico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ésteres do Colesterol/química , Ésteres do Colesterol/farmacologia , Diosgenina/análogos & derivados , Diosgenina/química , Diosgenina/farmacologia , Dissacarídeos/química , Dissacarídeos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Glucosídeos/química , Glucosídeos/farmacologia , Glicolipídeos/química , Glicolipídeos/farmacologia , Células HEK293 , Humanos , Indanos/química , Indanos/farmacologia , Micelas , Octoxinol/química , Octoxinol/farmacologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula , Xantenos/química , Xantenos/farmacologia
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