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1.
Proc Natl Acad Sci U S A ; 117(38): 23539-23547, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32907940

RESUMO

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.


Assuntos
RNA Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina
2.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641477

RESUMO

Positive-strand RNA [(+)RNA] viruses are important pathogens of humans, animals, and plants and replicate inside host cells by coopting numerous host factors and subcellular membranes. To gain insights into the assembly of viral replicase complexes (VRCs) and dissect the roles of various lipids and coopted host factors, we have reconstituted Tomato bushy stunt virus (TBSV) replicase using artificial giant unilamellar vesicles (GUVs). We demonstrate that reconstitution of VRCs on GUVs with endoplasmic reticulum (ER)-like phospholipid composition results in a complete cycle of replication and asymmetrical RNA synthesis, which is a hallmark of (+)RNA viruses. TBSV VRCs assembled on GUVs provide significant protection of the double-stranded RNA (dsRNA) replication intermediate against the dsRNA-specific RNase III. The lipid compositions of GUVs have pronounced effects on in vitro TBSV replication, including (-) and (+)RNA synthesis. The GUV-based assay has led to the discovery of the critical role of phosphatidylserine in TBSV replication and a novel role for phosphatidylethanolamine in asymmetrical (+)RNA synthesis. The GUV-based assay also showed stimulatory effects by phosphatidylinositol-3-phosphate [PI(3)P] and ergosterol on TBSV replication. We demonstrate that eEF1A and Hsp70 coopted replicase assembly factors, Vps34 phosphatidylinositol 3-kinase (PI3K) and the membrane-bending ESCRT factors, are required for reconstitution of the active TBSV VRCs in GUVs, further supporting that the novel GUV-based in vitro approach recapitulates critical steps and involves essential coopted cellular factors of the TBSV replication process. Taken together, this novel GUV assay will be highly suitable to dissect the functions of viral and cellular factors in TBSV replication.IMPORTANCE Understanding the mechanism of replication of positive-strand RNA viruses, which are major pathogens of plants, animals, and humans, can lead to new targets for antiviral interventions. These viruses subvert intracellular membranes for virus replication and coopt numerous host proteins, whose functions during virus replication are not yet completely defined. To dissect the roles of various host factors in Tomato bushy stunt virus (TBSV) replication, we have developed an artificial giant unilamellar vesicle (GUV)-based replication assay. The GUV-based in vitro approach recapitulates critical steps of the TBSV replication process. GUV-based reconstitution of the TBSV replicase revealed the need for a complex mixture of phospholipids, especially phosphatidylserine and phosphatidylethanolamine, in TBSV replication. The GUV-based approach will be useful to dissect the functions of essential coopted cellular factors.


Assuntos
RNA de Cadeia Dupla/genética , Tombusvirus/genética , Lipossomas Unilamelares/metabolismo , Proteínas Virais/genética , Bioensaio , Linhagem Celular , Retículo Endoplasmático/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ergosterol/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Lipossomas Unilamelares/química , Proteínas Virais/metabolismo , Replicação Viral
3.
Proc Natl Acad Sci U S A ; 117(28): 16401-16408, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601215

RESUMO

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.


Assuntos
Retículo Endoplasmático/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Células HeLa , Humanos , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Receptores de LDL/metabolismo
4.
PLoS Comput Biol ; 16(7): e1007996, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32667909

RESUMO

Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Potenciais da Membrana , Mitocôndrias/metabolismo , Vasoconstrição , Trifosfato de Adenosina/química , Cálcio/química , Fosfatos de Cálcio/química , Córtex Cerebral/fisiopatologia , Circulação Cerebrovascular , Citosol/química , Retículo Endoplasmático/química , Substância Cinzenta/fisiopatologia , Humanos , Modelos Teóricos , Acoplamento Neurovascular , Oscilometria , Oxigênio/química , Fosforilação , ATPases Translocadoras de Prótons/química , Acidente Vascular Cerebral/fisiopatologia
5.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
6.
Nucleic Acids Res ; 48(W1): W239-W243, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32421834

RESUMO

Recent evidences suggest that the localization of mRNAs near the subcellular compartment of the translated proteins is a more robust cellular tool, which optimizes protein expression, post-transcriptionally. Retention of mRNA in the nucleus can regulate the amount of protein translated from each mRNA, thus allowing a tight temporal regulation of translation or buffering of protein levels from bursty transcription. Besides, mRNA localization performs a variety of additional roles like long-distance signaling, facilitating assembly of protein complexes and coordination of developmental processes. Here, we describe a novel machine-learning based tool, mRNALoc, to predict five sub-cellular locations of eukaryotic mRNAs using cDNA/mRNA sequences. During five fold cross-validations, the maximum overall accuracy was 65.19, 75.36, 67.10, 99.70 and 73.59% for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. Assessment on independent datasets revealed the prediction accuracies of 58.10, 69.23, 64.55, 96.88 and 69.35% for extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The corresponding values of AUC were 0.76, 0.75, 0.70, 0.98 and 0.74 for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The mRNALoc standalone software and web-server are freely available for academic use under GNU GPL at http://proteininformatics.org/mkumar/mrnaloc.


Assuntos
RNA Mensageiro/análise , Software , Máquina de Vetores de Suporte , Núcleo Celular/química , Simulação por Computador , Citoplasma/química , Retículo Endoplasmático/química , Mitocôndrias/química , RNA Mensageiro/química , Análise de Sequência de RNA
7.
Science ; 369(6502): 433-436, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32439656

RESUMO

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.


Assuntos
Retículo Endoplasmático/química , Membranas Intracelulares/química , Proteínas de Membrana/química , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
9.
Chem Commun (Camb) ; 56(25): 3657-3660, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32108200

RESUMO

We report blue- and green-emitting two-photon probes derived from naphthalene and fluorene derivatives (as fluorophores) and an endoplasmic reticulum (ER) retrieval peptide (KDEL; as an ER-targeting moiety) that can detect the ER in a live cell by both one-photon and two-photon microscopy (TPM) and in a live tissue by TPM.


Assuntos
Retículo Endoplasmático/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica , Fótons , Fluorenos/química , Células HeLa , Humanos , Estrutura Molecular , Naftalenos/química , Imagem Óptica , Peptídeos/química
10.
Methods Mol Biol ; 2102: 531-555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989575

RESUMO

Endoplasmic reticulum (ER) stress is one of the major mechanisms underlying the etiology of multiple diseases and drug-induced toxicity. Gaussia luciferase (Gluc) is a naturally secreted protein that has been used as a reporter for the secretory pathway of ER to enable efficient and real-time monitoring of the ER function. The Gluc assay has been widely used and optimized in various labs. In this chapter, we provide an example of the application of the Gluc assay by establishing a stable cell line expressing both Gluc and firefly luciferase (Fluc) to study ER stress in liver cells. We describe the detailed procedures used in our laboratory for Gluc- and Fluc-containing lentivirus production and titration, for establishing a HepG2-based stable cell line through lentivirus transduction and the validation process. In addition, we provide an example of using the established stable cell line to investigate ER stress.


Assuntos
Técnicas de Cultura de Células/métodos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Hepatócitos/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferases/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Copépodes , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Genes Reporter , Células Hep G2 , Hepatócitos/química , Humanos , Lentivirus/genética , Luciferases/química , Luciferases/genética , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Fluxo de Trabalho
11.
Biochemistry ; 59(3): 260-269, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31718172

RESUMO

Most proteins are specifically localized in membrane-encapsulated organelles or non-membrane-bound compartments. The subcellular localization of proteins facilitates their functions and integration into functional networks; therefore, protein localization is tightly regulated in diverse biological contexts. However, protein localization has been mainly analyzed through immunohistochemistry or the fractionation of subcellular compartments, each of which has major drawbacks. Immunohistochemistry can examine only a handful of proteins at a time, and fractionation inevitably relies on the lysis of cells, which disrupts native cellular conditions. Recently, an engineered ascorbate peroxidase (APEX)-based proximity labeling technique combined with mass spectrometry was developed, which allows for temporally and spatially resolved proteomic mapping. In the presence of H2O2, engineered APEX oxidizes biotin-phenols into biotin-phenoxyl radicals, and these short-lived radicals biotinylate electron-rich amino acids within a radius of several nanometers. Biotinylated proteins are subsequently enriched by streptavidin and identified by mass spectrometry. This permits the sensitive and efficient labeling of proximal proteins around locally expressed APEX. Through the targeted expression of APEX in the subcellular region of interest, proteomic profiling of submitochondrial spaces, the outer mitochondrial membrane, the endoplasmic reticulum (ER)-mitochondrial contact, and the ER membrane has been performed. Furthermore, this method has been modified to define interaction networks in the vicinity of target proteins and has also been applied to analyze the spatial transcriptome. In this Perspective, we provide an outline of this newly developed technique and discuss its potential applications to address diverse biological questions.


Assuntos
Aminoácidos/química , Ascorbato Peroxidases/química , Mitocôndrias/genética , Transcriptoma/genética , Aminoácidos/genética , Ascorbato Peroxidases/genética , Biotina/química , Biotina/genética , Biotinilação/efeitos dos fármacos , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Radicais Livres/química , Perfilação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/química , Espectrometria de Massas , Mitocôndrias/química , Fenóis/química , Engenharia de Proteínas/tendências , Proteômica/tendências , Coloração e Rotulagem/métodos , Estreptavidina/química
12.
Artigo em Inglês | MEDLINE | ID: mdl-31146038

RESUMO

The turnover of phospholipids plays an essential role in membrane lipid homeostasis by impacting both lipid head group and acyl chain composition. This review focusses on the degradation and acyl chain remodeling of the major phospholipid classes present in the ER membrane of the reference eukaryote Saccharomyces cerevisiae, i.e. phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Phospholipid turnover reactions are introduced, and the occurrence and important functions of phospholipid remodeling in higher eukaryotes are briefly summarized. After presenting an inventory of established mechanisms of phospholipid acyl chain exchange, current knowledge of phospholipid degradation and remodeling by phospholipases and acyltransferases localized to the yeast ER is summarized. PC is subject to the PC deacylation-reacylation remodeling pathway (PC-DRP) involving a phospholipase B, the recently identified glycerophosphocholine acyltransferase Gpc1p, and the broad specificity acyltransferase Ale1p. PI is post-synthetically enriched in C18:0 acyl chains by remodeling reactions involving Cst26p. PE may undergo turnover by the phospholipid: diacylglycerol acyltransferase Lro1p as first step in acyl chain remodeling. Clues as to the functions of phospholipid acyl chain remodeling are discussed.


Assuntos
Retículo Endoplasmático/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acilação , Animais , Retículo Endoplasmático/química , Humanos , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/análise , Fosfatidilinositóis/metabolismo , Fosfolipídeos/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868591

RESUMO

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.


Assuntos
Proteínas de Caenorhabditis elegans/química , Manose/química , Proteínas de Membrana/química , Dobramento de Proteína , Receptores de Superfície Celular/química , Trombospondinas/química , Animais , Arginina/química , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/ultraestrutura , Cisteína/química , Dissulfetos/química , Drosophila melanogaster/química , Drosophila melanogaster/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Glicosilação , Ligação de Hidrogênio , Manose/genética , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/ultraestrutura , Trombospondinas/genética , Triptofano/química , Triptofano/genética
14.
Channels (Austin) ; 13(1): 483-497, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31679457

RESUMO

KCNE ß-subunits play critical roles in modulating cardiac voltage-gated potassium channels. Among them, KCNE1 associates with KCNQ1 channel to confer a slow-activated IKs current, while KCNE2 functions as a dominant negative modulator to suppress the current amplitude of KCNQ1. Any anomaly in these channels will lead to serious myocardial diseases, such as the long QT syndrome (LQTS). Trafficking defects of KCNE1 have been reported to account for the pathogenesis of LQT5. However, the molecular mechanisms underlying KCNE forward trafficking remain elusive. Here, we describe an arginine/lysine-based motif ([R/K](S)[R/K][R/K]) in the proximal C-terminus regulating the endoplasmic reticulum (ER) export of KCNE1 and KCNE2 in HEK293 cells. Notably, this motif is highly conserved in the KCNE family. Our results indicate that the forward trafficking of KCNE2 controlled by the motif (KSKR) is essential for suppressing the cell surface expression and current amplitude of KCNQ1. Unlike KCNE2, the motif (RSKK) in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1. Furthermore, truncations of the C-terminus did not reduce the apparent affinity of KCNE2 for KCNQ1, demonstrating that the rigid C-terminus of KCNE2 may not physically interact with KCNQ1. In contrast, the KCNE1 C-terminus is critical for its interaction with KCNQ1. These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2 membrane targeting and how they coassemble with KCNQ1 to regulate the channels activity.


Assuntos
Retículo Endoplasmático/metabolismo , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Motivos de Aminoácidos , Arginina/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Lisina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Transporte Proteico
15.
Biochem J ; 476(21): 3241-3260, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31696206

RESUMO

The tubular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions. Two classes of the conserved ER membrane proteins, atlastins and lunapark, have been shown to reside at the three-way junctions so far and be involved in the generation and stabilization of the three-way junctions. In this study, we report TMCC3 (transmembrane and coiled-coil domain family 3), a member of the TEX28 family, as another ER membrane protein that resides at the three-way junctions in mammalian cells. When the TEX28 family members were transfected into U2OS cells, TMCC3 specifically localized at the three-way junctions in the peripheral ER. TMCC3 bound to atlastins through the C-terminal transmembrane domains. A TMCC3 mutant lacking the N-terminal coiled-coil domain abolished localization to the three-way junctions, suggesting that TMCC3 localized independently of binding to atlastins. TMCC3 knockdown caused a decrease in the number of three-way junctions and expansion of ER sheets, leading to a reduction of the tubular ER network in U2OS cells. The TMCC3 knockdown phenotype was partially rescued by the overexpression of atlastin-2, suggesting that TMCC3 knockdown would decrease the activity of atlastins. These results indicate that TMCC3 localizes at the three-way junctions for the proper tubular ER network.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Domínios Proteicos , Transporte Proteico
16.
ACS Sens ; 4(11): 2858-2863, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31617349

RESUMO

Endoplasmic reticulum-thioflavin T (ER-ThT), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.


Assuntos
Benzotiazóis/metabolismo , Técnicas Biossensoriais , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Tapsigargina/farmacologia , Benzotiazóis/análise , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Espectrometria de Fluorescência , Tapsigargina/química
17.
Bioorg Chem ; 92: 103295, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31546206

RESUMO

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.


Assuntos
Corantes Fluorescentes/química , Hidrazonas/química , Saccharomyces cerevisiae/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Hidrazonas/síntese química , Hidrazonas/farmacologia , Microscopia de Fluorescência , Estrutura Molecular , Saccharomyces cerevisiae/química , Relação Estrutura-Atividade
18.
Nat Commun ; 10(1): 3400, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363088

RESUMO

Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.


Assuntos
Compostos de Boro/química , Rastreamento de Células/métodos , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , Linhagem Celular Tumoral , Rastreamento de Células/instrumentação , Células/química , Células/citologia , Células/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/instrumentação
19.
Prog Lipid Res ; 75: 100990, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31442527

RESUMO

Environmental stresses cause membrane damage in terrestrial plants. Studies on the lipids obtained from these plants are required to understand their adaptation to climate change. Recently, a number of plant leaf lipidomic studies converged on the topic of chloroplastic glycerolipid remodeling and triacylglycerol production. In this review, we show that among various abiotic stresses, plant leaves under heat stress specifically increase the levels of galactolipids containing linoleate (18:2) in chloroplasts; phospholipids containing palmitate (16:0), stearate (18:0), and oleate (18:1) in the endoplasmic reticulum and plasma membrane; and triacylglycerol containing α-linolenate (18:3) and hexadecatrienoic acid (16:3) as lipid droplets in the leaves of Arabidopsis thaliana. Recent studies have proposed responsible genes for the lipid remodeling under heat stress, highlighting the importance of the catabolic process of chloroplastic monogalactosyldiacylglycerol. This review comprehensively describes glycerolipid compositional changes in plant leaves under heat stress detected by lipidomic analyses and compares them with those under other abiotic stresses. We will discuss the physiological significance underlying the observed lipid metabolism under heat stress. Detailed knowledge about plant lipid remodeling can aid in the development of solutions to deal with the consequences of climate change, including global warming.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Membrana Celular/química , Glicolipídeos/análise , Folhas de Planta/química , Arabidopsis/química , Cloroplastos/química , Retículo Endoplasmático/química , Resposta ao Choque Térmico , Lipidômica/métodos , Lipídeos de Membrana/química
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