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1.
Nat Commun ; 12(1): 5963, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645814

RESUMO

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.


Assuntos
Adenosina Trifosfatases/química , Lisofosfolipídeos/química , Proteínas de Membrana Transportadoras/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membrana Celular/química , Membrana Celular/enzimologia , Clonagem Molecular , Microscopia Crioeletrônica , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
2.
Elife ; 102021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543184

RESUMO

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Axônios/enzimologia , Membrana Celular/enzimologia , Hipocampo/enzimologia , Vesículas Sinápticas/enzimologia , Sinaptotagminas/metabolismo , Animais , Axônios/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas , Exocitose , Hipocampo/ultraestrutura , Lipoilação , Camundongos Knockout , Simulação de Acoplamento Molecular , Plasticidade Neuronal , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteólise , Ratos Sprague-Dawley , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura , Sinaptotagminas/genética , Fatores de Tempo
3.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575984

RESUMO

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell's direct coupling with conformational coupling to catalyze cation transport across the membrane.


Assuntos
Catálise , Difosfatos/química , Pirofosfatase Inorgânica/química , Canais Iônicos/química , Membrana Celular/enzimologia , Dimerização , Hidrólise , Canais Iônicos/genética , Transporte de Íons/genética , Cinética , Potássio/química , Prótons , Pirofosfatases
4.
Nat Commun ; 12(1): 5709, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588453

RESUMO

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.


Assuntos
Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Potássio/metabolismo , Sítios de Ligação/genética , Cátions Monovalentes/metabolismo , Membrana Celular/enzimologia , Microscopia Crioeletrônica , Cristalização , Ensaios Enzimáticos , Mucosa Gástrica/citologia , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/isolamento & purificação , ATPase Trocadora de Hidrogênio-Potássio/ultraestrutura , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato/genética
5.
J Biol Chem ; 297(4): 101227, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34562451

RESUMO

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.


Assuntos
Membrana Celular/enzimologia , Precursores Enzimáticos/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Serina Endopeptidases/metabolismo , Animais , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Precursores Enzimáticos/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Domínios Proteicos , Transporte Proteico/genética , Serina Endopeptidases/genética
6.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443697

RESUMO

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled "Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents" edited by Mary K. Phillips-Jones.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/enzimologia , Descoberta de Drogas , Histidina Quinase/química , Histidina Quinase/metabolismo , Transdução de Sinais , Ligantes , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacos
7.
Cells ; 10(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34440712

RESUMO

Fatty acids are important biological components, yet the metabolism of fatty acids in microalgae is not clearly understood. Previous studies found that Chlamydomonas reinhardtii, the model microalga, incorporates exogenously added fatty acids but metabolizes them differently from animals and yeast. Furthermore, a recent metabolic flux analysis found that the majority of lipid turnover in C. reinhardtii is the recycling of acyl chains from and to membranes, rather than ß -oxidation. This indicates that for the alga, the maintenance of existing acyl chains may be more valuable than their breakdown for energy. To gain cell-biological knowledge of fatty acid metabolism in C. reinhardtii, we conducted microscopy analysis with fluorescent probes. First, we found that CAT1 (catalase isoform 1) is in the peroxisomes while CAT2 (catalase isoform 2) is localized in the endoplasmic reticulum, indicating the alga is capable of detoxifying hydrogen peroxide that would be produced during ß-oxidation in the peroxisomes. Second, we compared the localization of exogenously added FL-C16 (fluorescently labelled palmitic acid) with fluorescently marked endosomes, mitochondria, peroxisomes, lysosomes, and lipid droplets. We found that exogenously added FL-C16 are incorporated and compartmentalized via a non-endocytic route within 10 min. However, the fluorescence signals from FL-C16 did not colocalize with any marked organelles, including peroxisomes. During triacylglycerol accumulation, the fluorescence signals from FL-C16 were localized in lipid droplets. These results support the idea that membrane turnover is favored over ß-oxidation in C. reinhardtii. The knowledge gained in these analyses would aid further studies of the fatty acid metabolism.


Assuntos
Catalase/metabolismo , Membrana Celular/enzimologia , Chlamydomonas reinhardtii/enzimologia , Retículo Endoplasmático/enzimologia , Gotículas Lipídicas/metabolismo , Ácido Palmítico/metabolismo , Peroxissomos/enzimologia , Proteínas de Plantas/metabolismo , Catalase/genética , Membrana Celular/genética , Chlamydomonas reinhardtii/genética , Peróxido de Hidrogênio/metabolismo , Isoenzimas , Microscopia de Fluorescência , Oxirredução , Proteínas de Plantas/genética , Fatores de Tempo
8.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199294

RESUMO

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.


Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Membrana Celular/enzimologia , Temperatura Baixa , Proteínas Quinases Ativadas por Mitógeno/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Congelamento , Cinética , Fluidez de Membrana , Biossíntese de Proteínas , Transcrição Genética
9.
Toxins (Basel) ; 13(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918753

RESUMO

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.


Assuntos
Toxinas Botulínicas/metabolismo , Catepsina B/metabolismo , Membrana Celular/enzimologia , Clostridium botulinum/metabolismo , Endocitose , Lisossomos/enzimologia , Animais , Catepsina B/genética , Membrana Celular/microbiologia , Clostridium botulinum/patogenicidade , Cães , Exocitose , Interações Hospedeiro-Patógeno , Lisossomos/genética , Lisossomos/microbiologia , Células Madin Darby de Rim Canino , Esfingomielina Fosfodiesterase/metabolismo
10.
Arch Biochem Biophys ; 703: 108870, 2021 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-33831357

RESUMO

Membrane-associated proteins carry out a wide range of essential cellular functions but the structural characterization needed to understand these functions is dramatically underrepresented in the Protein Data Bank. Producing a soluble, stable and active form of a membrane-associated protein presents formidable challenges, as evidenced by the variety of approaches that have been attempted with a multitude of different membrane proteins to achieve this goal. Aspartate N-acetyltransferase (ANAT) is a membrane-anchored enzyme that performs a critical function, the synthesis of N-acetyl-l-aspartate (NAA), the second most abundant amino acid in the brain. This amino acid is a precursor for a neurotransmitter, and alterations in brain NAA levels have been implicated as a causative effect in Canavan disease and has been suggested to be involved in other neurological disorders. Numerous prior attempts have failed to produce a soluble form of ANAT that is amenable for functional and structural investigations. Through the application of a range of different approaches, including fusion partner constructs, linker modifications, membrane-anchor modifications, and domain truncations, a highly soluble, stable and fully active form of ANAT has now been obtained. Producing this modified enzyme form will accelerate studies aimed at structural characterization and structure-guided inhibitor development.


Assuntos
Acetiltransferases/genética , Acetiltransferases/metabolismo , Biocatálise , Membrana Celular/enzimologia , Engenharia de Proteínas , Acetiltransferases/química , Sequência de Aminoácidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Linhagem Celular , Humanos , Domínios Proteicos , Solubilidade
11.
Elife ; 102021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33825682

RESUMO

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, amyotrophic lateral sclerosis (ALS). Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid-induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association-mediated toxic aggregation and survival time scale after ALS diagnosis.


Assuntos
Esclerose Amiotrófica Lateral/enzimologia , Membrana Celular/enzimologia , Neurônios/enzimologia , Superóxido Dismutase-1/metabolismo , Zinco/metabolismo , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/patologia , Cobre/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neurônios/patologia , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética
12.
Biochem Biophys Res Commun ; 550: 120-126, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33691198

RESUMO

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.


Assuntos
Padrões Moleculares Associados a Patógenos/antagonistas & inibidores , Imunidade Vegetal , Ralstonia solanacearum/enzimologia , Solanum tuberosum/imunologia , Solanum tuberosum/microbiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Virulência , Motivos de Aminoácidos , Biocatálise , Morte Celular , Membrana Celular/enzimologia , Cisteína/metabolismo , Flagelina/química , Flagelina/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Ralstonia solanacearum/genética , Ubiquitina-Proteína Ligases/química , Virulência/genética
13.
Dev Cell ; 56(7): 933-948, 2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33761322

RESUMO

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.


Assuntos
Parede Celular/metabolismo , Células Vegetais/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Parede Celular/química , Parede Celular/ultraestrutura , Endocitose , Retículo Endoplasmático/metabolismo , Glucosiltransferases/metabolismo , Complexo de Golgi/metabolismo , Homeostase , Células Vegetais/enzimologia , Células Vegetais/ultraestrutura , Polissacarídeos/biossíntese
14.
J Biol Chem ; 296: 100470, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33639165

RESUMO

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.


Assuntos
Antivirais/farmacologia , Carbamatos/farmacologia , Dioxanos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glicoesfingolipídeos/antagonistas & inibidores , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Pirrolidinas/farmacologia , Quinuclidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/síntese química , COVID-19/tratamento farmacológico , COVID-19/enzimologia , COVID-19/virologia , Carbamatos/síntese química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/virologia , Chlorocebus aethiops , Ensaios Clínicos Fase III como Assunto , Dioxanos/síntese química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/biossíntese , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Pirrolidinas/síntese química , Quinuclidinas/síntese química , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/metabolismo , Transdução de Sinais , Células Vero , Replicação Viral/efeitos dos fármacos
15.
Plant Sci ; 304: 110802, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568301

RESUMO

Arabidopsis Toxicos en Levadura (ATL) proteins compose a subfamily of E3 ubiquitin ligases and play major roles in regulating plant growth, cold, drought, oxidative stresses response and pathogen defense in plants. However, the role in enhancing salt tolerance has not been reported to date. Here, we cloned a novel RING-H2 type E3 ubiquitin ligase gene, named IbATL38, from sweetpotato cultivar Lushu 3. This gene was highly expressed in the leaves of sweetpotato and strongly induced by NaCl and abscisic acid (ABA). This IbATL38 was localized to nucleus and plasm membrane and possessed E3 ubiquitin ligase activity. Overexpression of IbATL38 in Arabidopsis significantly enhanced salt tolerance, along with inducible expression of a series of stress-responsive genes and prominently decrease of H2O2 content. These results suggest that IbATL38 as a novel E3 ubiquitin ligase may play an important role in salt stress response.


Assuntos
Ipomoea batatas/enzimologia , Proteínas de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Arabidopsis , Membrana Celular/enzimologia , Núcleo Celular/enzimologia , Clonagem Molecular , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Tolerância ao Sal , Análise de Sequência , Análise de Sequência de DNA , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia
16.
Commun Biol ; 4(1): 159, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542467

RESUMO

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.


Assuntos
Membrana Celular/enzimologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Quinurenina 3-Mono-Oxigenase/metabolismo , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Células HEK293 , Humanos , Quinurenina 3-Mono-Oxigenase/química , Quinurenina 3-Mono-Oxigenase/genética , Ligantes , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Ratos , Relação Estrutura-Atividade
17.
Bioelectrochemistry ; 138: 107683, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33421898

RESUMO

Dissimilatory metal-reducing bacteria (DMRB) have a variety of c-type cytochromes (OM c-cyts) intercalated in their outer membrane, and this structure serves as the physiological basis for DMRB to carry out the extracellular electron transfer processes. Using Geobacter sulfurreducens as a model DMRB, we demonstrated that visible-light illumination could alter the electronic state of OM c-cyts from the ground state to the excited state in vivo. The existence of excited-state OM c-cyts in vivo was confirmed by spectroscopy. More importantly, excited-state OM c-cyts had a more negative potential compared to their ground-state counterparts, conferring DMRB with an extra pathway to transfer electrons to semi-conductive electron acceptors. To demonstrate this, using a TiO2-coated electrode as an electron acceptor, we showed that G. sulfurreducens could directly utilise the conduction band of TiO2 as an electron acceptor under visible-light illumination (λ > 420 nm) without causing TiO2 charge separation. When G. sulfurreducens was subject to visible-light illumination, the rate of extracellular electron transfer (EET) to TiO2 accelerated by over 8-fold compared to that observed under dark conditions. Results of additional electrochemical tests provided complementary evidence to support that G. sulfurreducens utilised excited-state OM c-cyts to enhance EET to TiO2.


Assuntos
Membrana Celular/enzimologia , Citocromos c/metabolismo , Espaço Extracelular/metabolismo , Espaço Extracelular/efeitos da radiação , Geobacter/citologia , Geobacter/metabolismo , Luz , Transporte de Elétrons/efeitos da radiação , Geobacter/efeitos da radiação , Titânio/química
18.
Curr Genet ; 67(2): 255-262, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33388852

RESUMO

Polarized growth is required in eukaryotic cells for processes such as cell division, morphogenesis and motility, which involve conserved and interconnected signalling pathways controlling cell cycle progression, cytoskeleton reorganization and secretory pathway functioning. While many of the factors involved in polarized growth are known, it is not yet clear how they are coordinated both spatially and temporally. Several lines of evidence point to the important role of lipid flippases in polarized growth events. Lipid flippases, which mainly belong to the P4 subfamily of P-type ATPases, are active transporters that move different lipids to the cytosolic side of biological membranes at the expense of ATP. The involvement of the Saccharomyces cerevisiae plasma membrane P4 ATPases Dnf1p and Dnf2p in polarized growth and their activation by kinase phosphorylation were established some years ago. However, these two proteins do not seem to be responsible for the phosphatidylserine internalization required for early recruitment of proteins to the plasma membrane during yeast mating and budding. In a recent publication, we demonstrated that the Golgi-localized P4 ATPase Dnf3p has a preference for PS as a substrate, can reach the plasma membrane in a cell cycle-dependent manner, and is regulated by the same kinases that activate Dnf1p and Dnf2p. This finding solves a long-lasting enigma in the field of lipid flippases and suggests that tight and heavily coordinated spatiotemporal control of lipid translocation at the plasma membrane is important for proper polarized growth.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , ATPases do Tipo-P/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Transporte Biológico/genética , Membrana Celular/enzimologia , Proliferação de Células/genética , Células Eucarióticas/enzimologia , Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
19.
PLoS One ; 16(1): e0245679, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33507968

RESUMO

The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Membrana Celular/enzimologia , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Planta ; 253(1): 10, 2021 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-33389194

RESUMO

MAIN CONCLUSION: The plasma membrane H+-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H+-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced Vmax, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H+ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H+-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5 kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H+-ATPase redox regulation in this process is discussed.


Assuntos
Diamida , Ervilhas , Raízes de Plantas , ATPases Translocadoras de Prótons , Membrana Celular/enzimologia , Diamida/farmacologia , Ervilhas/enzimologia , Raízes de Plantas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia
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