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1.
PLoS Biol ; 17(10): e3000512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658248

RESUMO

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.


Assuntos
Arrestinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Nucleosídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tiamina/metabolismo , Arrestinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Endocitose/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Proteínas de Transporte de Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
2.
Biomed Res Int ; 2019: 5496197, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583245

RESUMO

Background and Aims: Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). Methods: Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. Results: Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [125I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [125I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. Conclusion: Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.


Assuntos
Fígado/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Membrana Celular/genética , Endocitose/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/patologia , RNA Mensageiro/genética , Ratos , Transdução de Sinais/genética
3.
PLoS Genet ; 15(10): e1008263, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626625

RESUMO

In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.


Assuntos
Bacillus subtilis/genética , Integrases/genética , Proteínas de Membrana Transportadoras/genética , Fator sigma/genética , Bacillus subtilis/crescimento & desenvolvimento , Membrana Celular/genética , Parede Celular/genética , Dano ao DNA/genética , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Óperon/genética , Regiões Promotoras Genéticas , Regulon/genética
4.
PLoS Biol ; 17(10): e3000475, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584943

RESUMO

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.


Assuntos
Azidas/química , Reagentes para Ligações Cruzadas/química , Proteínas do Citoesqueleto/química , Citoesqueleto/metabolismo , Membranas Intracelulares/metabolismo , Fenilalanina/análogos & derivados , Proteínas de Protozoários/química , Toxoplasma/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Expressão Gênica , Membranas Intracelulares/ultraestrutura , Fenilalanina/química , Processos Fotoquímicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/ultraestrutura , Raios Ultravioleta
5.
J Agric Food Chem ; 67(49): 13673-13683, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31617717

RESUMO

Because Monascus pigments (MPs) predominantly accumulate in the cytoplasm during submerged fermentation, many biotechnologies are applied to enhance the production of extracellular MPs (exMPs) to reduce the downstream processing costs. In this study, the genes monascus_7017 and monascus_8018, identified as ERG4 genes, were knocked out to disrupt the ergosterol biosynthetic pathway and enhance the production of exMPs in Monascus purpureus LQ-6. Double-deletion of EGR4 in M. purpureus LQ-6 reduced ergosterol concentration by 57.14% and enhanced exMP production 2.06-fold. In addition, integrated transcriptomic and proteomic analyses were performed to elucidate the transmembrane secretion mechanism of exMPs based on the relationship between ergosterol synthesis and membrane permeability, which revealed that several metabolic pathways were noticeably dynamic, including fatty acid degradation, amino acid metabolism, energy metabolism, carbohydrate metabolism, and transport. These findings therefore clarified the secretion mechanism of exMPs and provide a novel strategy for further enhancement of exMP production in submerged fermentation.


Assuntos
Membrana Celular/metabolismo , Ergosterol/biossíntese , Monascus/metabolismo , Pigmentos Biológicos/biossíntese , Vias Biossintéticas , Membrana Celular/genética , Permeabilidade da Membrana Celular , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Monascus/genética
6.
J Integr Bioinform ; 16(3)2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31560649

RESUMO

For more than one decade, CELLmicrocosmos tools are being developed. Here, we discus some of the technical and administrative hurdles to keep a software suite running so many years. The tools were being developed during a number of student projects and theses, whereas main developers refactored and maintained the code over the years. The focus of this publication is laid on two Java-based Open Source Software frameworks. Firstly, the CellExplorer with the PathwayIntegration combines the mesoscopic and the functional level by mapping biological networks onto cell components using database integration. Secondly, the MembraneEditor enables users to generate membranes of different lipid and protein compositions using the PDB format. Technicalities will be discussed as well as the historical development of these tools with a special focus on group-based development. In this way, university-associated developers of Integrative Bioinformatics applications should be inspired to go similar ways. All tools discussed in this publication can be downloaded and installed from https://www.CELLmicrocosmos.org.


Assuntos
Membrana Celular , Biologia Computacional/história , Gráficos por Computador/história , Modelos Biológicos , Linguagens de Programação , Membrana Celular/genética , Membrana Celular/metabolismo , História do Século XX , História do Século XXI , Humanos
7.
Biomed Res Int ; 2019: 3534943, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396528

RESUMO

Apart from their significance in the protection against stress conditions, the plant cell membranes are essential for proper development of the diverse surface structures formed on aerial plant organs. In addition, we signal that membrane remodeling and integrity are function of some of causal physiological and enzymological aspects such as the MDA, the ion leakage and also the monitoring of some phytozymes involved in lipid and cellulose metabolisms. Those last ones are related to the membrane structure (lipases and cellulases), that were assessed in durum wheat dehydrin transgenic context (YS, K1-K2, DH2, and DH4), proline metabolic mutant (P5CS1-4) per comparison with the wild-type plant (Wt). We report also the docking data reinforcing the fact that the membrane integrity seems to be function of causal enzymological behaviors, through the molecular dynamic investigation resulting from the dehydrin-phytozyme interactions and also from the inhibition effect of the durum wheat LTP4 on the lipase activity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Plantas Geneticamente Modificadas , Estresse Salino , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/enzimologia , Membrana Celular/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Triticum
8.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31400094

RESUMO

Mycobacteria, including the infamous pathogen Mycobacterium tuberculosis, are high-GC Gram-positive bacteria with a distinctive cell envelope. Although there is a typical inner membrane, the mycobacterial cell envelope is unusual in having its peptidoglycan layer connected to a polymer of arabinogalactan, which in turn is covalently attached to long-chain mycolic acids that help form a highly impermeable mycobacterial outer membrane. This complex double-membrane, or diderm, cell envelope imparts mycobacteria with unique requirements for protein export into and across the cell envelope for secretion into the extracellular environment. In this article, we review the four protein export pathways known to exist in mycobacteria: two conserved systems that exist in all types of bacteria (the Sec and Tat pathways) and two specialized systems that exist in mycobacteria, corynebacteria, and a subset of low-GC Gram-positive bacteria (the SecA2 and type VII secretion pathways). We describe the progress made over the past 15 years in understanding each of these mycobacterial export pathways, and we highlight the need for research to understand the specific steps of protein export across the mycobacterial outer membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Mycobacterium tuberculosis/metabolismo , Animais , Proteínas de Bactérias/genética , Membrana Celular/genética , Parede Celular/genética , Humanos , Mycobacterium tuberculosis/genética , Transporte Proteico , Tuberculose/microbiologia
9.
Mol Carcinog ; 58(11): 2077-2090, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31411358

RESUMO

The plasma membrane (PM) is considered as a major druggable site. More than 50% of the existing drugs target PM proteins. In the wake of emerging data indicating a key role of estrogens in prostate cancer (PCa) pathogenesis, the study was undertaken to explore whether the estrogen binding sites exist on the PM and if such sites are functionally relevant in PCa. Estradiol (E2) binding to the PM was detected in androgen-dependent (LNCaP), androgen-independent (PC3, DU145) PCa cell lines, nontumorigenic (RWPE1) prostate epithelial cell line, and rat prostate cells. Conventional estrogen receptors (nuclear estrogen receptors), known for their nuclear localization, were detected in the PM enriched extracts. This was indirectly confirmed by reduced localization of ERs on the PM of cells, silenced for the expression of their cognate genes. Further, unlike cell-permeable E2, stimulation with cell-impermeable estradiol (E2-BSA) did not induce proliferation in LNCaP cells. However, stimulation with E2-BSA led to alterations in the phosphorylation status of several kinases including GSK3 and AKT, along with the hyperphosphorylation of cytoskeletal proteins such as ß-actin and cytokeratin 8 in LNCaP. This was accompanied by epithelial-to-mesenchymal (EMT) features such as increased migration and invasion; higher vimentin expression, and a concomitant decrease in the E-cadherin expression. These effects were not observed in RWPE1 cells. Interestingly, cell-permeable E2 failed to induce EMT in PCa cells. This in vitro study is the first to suggest that the PM-initiated estrogen signaling contributes to higher invasiveness in PCa cells. Plasma membrane ERs may act as novel targets for PCa therapeutics.


Assuntos
Androgênios/metabolismo , Membrana Celular/genética , Estrogênios/metabolismo , Neoplasias da Próstata/genética , Animais , Caderinas/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Transição Epitelial-Mesenquimal/genética , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Humanos , Queratina-8/genética , Masculino , Camundongos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Ratos , Transdução de Sinais
10.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373268

RESUMO

In bacteria, the Sec translocase mediates the translocation of proteins into and across the cytoplasmic membrane. It consists of a protein conducting channel SecYEG, the ATP-dependent motor SecA, and the accessory SecDF complex. Here we discuss the function and structure of the Sec translocase.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/metabolismo , /metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Transporte Proteico , Canais de Translocação SEC/genética , /genética
11.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382643

RESUMO

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Lipídeos de Membrana/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/química , Proteínas de Arabidopsis/química , Membrana Celular/química , Membrana Celular/genética , Exocitose/genética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/genética , Tricomas/química , Tricomas/genética , Proteínas de Transporte Vesicular/química
12.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298206

RESUMO

The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells. The injection apparatus is composed of a baseplate on which is built a contractile tail tube/sheath complex. The inner tube, topped by the spike complex, is propelled outside of the cell by the contraction of the sheath. The injection system is anchored to the cell envelope and oriented towards the cell exterior by a trans-envelope complex. Effectors delivered by the T6SS are loaded within the inner tube or on the spike complex and can target prokaryotic and/or eukaryotic cells. Here we summarize the structure, assembly, and mechanism of action of the T6SS. We also review the function of effectors and their mode of recruitment and delivery.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VI/química , Sistemas de Secreção Tipo VI/metabolismo , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Transporte Proteico , Sistemas de Secreção Tipo VI/genética
13.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31267890

RESUMO

The highly cross-linked peptidoglycan represents the rigid layer of the bacterial envelope and protects bacteria from osmotic lysis. In Gram-positive bacteria, peptidoglycan also functions as a scaffold for the immobilization of capsular polysaccharide, wall teichoic acid (WTA), and surface proteins. This chapter captures recent development on the assembly of the envelope of Staphylococcus aureus including mechanisms accounting for immobilization of molecules to peptidoglycan as well as hydrolysis of peptidoglycan for the specific release of bound molecules, facilitation of protein secretion across the envelope and cell division. Peptidoglycan, WTA and capsular polysaccharide are directly synthesized onto undecaprenol. Surface proteins are anchored by Sortase A, a membrane-embedded transpeptidase that scans secreted polypeptides for the C-terminal LPXTG motif of sorting signals. The resulting acyl enzyme intermediate is resolved by lipid II, the undecaprenol-bound peptidoglycan precursor. While these pathways share membrane diffusible undecaprenol, assembly of these molecules occurs either at the cross-walls or the cell poles. In S. aureus, the cross-wall represents the site of de novo peptidoglycan synthesis which is eventually split to complete the cell cycle yielding newly divided daughter cells. Peptidoglycan synthesized at the cross-wall is initially devoid of WTA. Conversely, lipoteichoic acid (LTA) synthesis which does not require bactoprenol is seemingly restricted to septal membranes. Similarly, S. aureus distinguishes two types of surface protein precursors. Polypeptides with canonical signal peptides are deposited at the cell poles, whereas precursors with conserved YSIRK-GXXS motif signal peptides traffic to the cross-wall. A model for protein trafficking in the envelope and uneven distribution of teichoic acids is discussed.


Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/genética , Membrana Celular/genética , Parede Celular/genética , Parede Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Peptidoglicano/metabolismo , Transporte Proteico , Staphylococcus aureus/genética
14.
Elife ; 82019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31318332

RESUMO

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.


Assuntos
Membrana Celular/genética , Canais de Cloreto/genética , Cloretos/metabolismo , Ácidos/metabolismo , Animais , Ânions/metabolismo , Morte Celular/genética , Membrana Celular/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Genoma Humano/genética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Prótons
15.
Curr Microbiol ; 76(11): 1290-1297, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31321468

RESUMO

The Lipid A component of the outer membrane of Gram-negative bacteria is an integral part of the permeability barrier known as LPS, which actively prevents the uptake of bactericidal compounds. It is clinically very significant, as it is known to elicit a strong immune response in the humans, through the TLR4 complex. The Lipid A species are synthesized through a highly conserved multistep biosynthetic pathway. The final step is catalyzed by acyltransferases of the HtrB/MsbB family, which are members of a superfamily of enzymes, present in all domains of life with important roles to play in various biological processes. The investigation of a putative dual functioning enzyme which can add both laurate and myristate residues to the (Kdo)2-lipid IVA (precursor of Lipid A) would give a snapshot into the versatility of substrates that these enzymes catalyze. In this study we have cloned and purified to homogeneity, such a putative dual functional acyltransferase from Chlorobium tepidum, and attempted to study the enzyme in more details in terms of its sequence and structural aspects, as it lacks conserved residues with other enzymes of the same family.


Assuntos
Aciltransferases/química , Proteínas de Bactérias/química , Membrana Celular/enzimologia , Chlorobium/enzimologia , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorobium/química , Chlorobium/genética , Chlorobium/metabolismo , Glicolipídeos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lipídeo A/análogos & derivados , Lipídeo A/metabolismo , Filogenia , Alinhamento de Sequência
16.
Microbiol Spectr ; 7(4)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31322105

RESUMO

Dating back to the 1960s, initial studies on the staphylococcal cell wall were driven by the need to clarify the mode of action of the first antibiotics and the resistance mechanisms developed by the bacteria. During the following decades, the elucidation of the biosynthetic path and primary composition of staphylococcal cell walls was propelled by advances in microbial cell biology, specifically, the introduction of high-resolution analytical techniques and molecular genetic approaches. The field of staphylococcal cell wall gradually gained its own significance as the complexity of its chemical structure and involvement in numerous cellular processes became evident, namely its versatile role in host interactions, coordination of cell division and environmental stress signaling.This chapter includes an updated description of the anatomy of staphylococcal cell walls, paying particular attention to information from the last decade, under four headings: high-resolution analysis of the Staphylococcus aureus peptidoglycan; variations in peptidoglycan composition; genetic determinants and enzymes in cell wall synthesis; and complex functions of cell walls. The latest contributions to a more precise picture of the staphylococcal cell envelope were possible due to recently developed state-of-the-art microscopy and spectroscopy techniques and to a wide combination of -omics approaches, that are allowing to obtain a more integrative view of this highly dynamic structure.


Assuntos
Parede Celular/química , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/citologia , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Humanos , Peptidoglicano/química , Peptidoglicano/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
17.
Mol Cell ; 75(5): 1031-1042.e4, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31327636

RESUMO

Every bacterial population harbors a small subpopulation of so-called persisters that are transiently antibiotic tolerant. These persisters are associated with the recalcitrance of chronic infections because they can recolonize the host after antibiotic removal. Although several effectors have been described to induce persistence, persister cell awakening is poorly understood. We previously reported that the toxin HokB induces persistence via pore formation, resulting in membrane depolarization and ATP leakage. We now delineate mechanisms responsible for the awakening of HokB-induced persisters. We show that HokB dimerization by the oxidoreductase DsbA is essential for pore formation and peptide stability. Pores are disassembled via DsbC-mediated monomerization, which targets HokB for DegQ-mediated degradation. Finally, pore disassembly allows membrane repolarization by the electron transport chain, supporting cells to resume growth. These results provide a detailed view of both the formation and awakening of HokB-induced persister cells.


Assuntos
Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Proteólise , Serina Endopeptidases/metabolismo , Toxinas Bacterianas/genética , Membrana Celular/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Serina Endopeptidases/genética
18.
Elife ; 82019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31329101

RESUMO

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.


Assuntos
Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/tratamento farmacológico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Animais , Membrana Celular/genética , Membrana Celular/fisiologia , Polaridade Celular/genética , Humanos , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Piperidinas/farmacologia , Potássio/metabolismo , Tiazóis/farmacologia , Compostos de Tosil/farmacologia , Xenopus/genética
19.
Int J Mol Sci ; 20(15)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344783

RESUMO

Investigations of information dynamics in eukaryotic cells focus almost exclusively on heritable information in the genome. Gene networks are modeled as "central processors" that receive, analyze, and respond to intracellular and extracellular signals with the nucleus described as a cell's control center. Here, we present a model in which cellular information is a distributed system that includes non-genomic information processing in the cell membrane that may quantitatively exceed that of the genome. Within this model, the nucleus largely acts a source of macromolecules and processes information needed to synchronize their production with temporal variations in demand. However, the nucleus cannot produce microsecond responses to acute, life-threatening perturbations and cannot spatially resolve incoming signals or direct macromolecules to the cellular regions where they are needed. In contrast, the cell membrane, as the interface with its environment, can rapidly detect, process, and respond to external threats and opportunities through the large amounts of potential information encoded within the transmembrane ion gradient. Our model proposes environmental information is detected by specialized protein gates within ion-specific transmembrane channels. When the gate receives a specific environmental signal, the ion channel opens and the received information is communicated into the cell via flow of a specific ion species (i.e., K+, Na+, Cl-, Ca2+, Mg2+) along electrochemical gradients. The fluctuation of an ion concentration within the cytoplasm adjacent to the membrane channel can elicit an immediate, local response by altering the location and function of peripheral membrane proteins. Signals that affect a larger surface area of the cell membrane and/or persist over a prolonged time period will produce similarly cytoplasmic changes on larger spatial and time scales. We propose that as the amplitude, spatial extent, and duration of changes in cytoplasmic ion concentrations increase, the information can be communicated to the nucleus and other intracellular structure through ion flows along elements of the cytoskeleton to the centrosome (via microtubules) or proteins in the nuclear membrane (via microfilaments). These dynamics add spatial and temporal context to the more well-recognized information communication from the cell membrane to the nucleus following ligand binding to membrane receptors. Here, the signal is transmitted and amplified through transduction by the canonical molecular (e.g., Mitogen Activated Protein Kinases (MAPK) pathways. Cytoplasmic diffusion allows this information to be broadly distributed to intracellular organelles but at the cost of loss of spatial and temporal information also contained in ligand binding.


Assuntos
Comunicação Celular/genética , Membrana Celular/genética , Núcleo Celular/genética , Células Eucarióticas , Cálcio/metabolismo , Citoplasma/genética , Citoesqueleto/genética , Genoma/genética , Canais Iônicos/genética , Canais Iônicos/metabolismo , Íons/metabolismo , Transdução de Sinais/genética
20.
Nat Commun ; 10(1): 3358, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350400

RESUMO

The integration of ß-barrel proteins into the bacterial outer membrane (OM) is catalysed by the ß-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a ß-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli ß-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first ß-strand of a laterally 'open' form of the BamA ß-barrel forms a rigid interface with the C-terminal ß-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA ß-strands forms weaker, conformationally heterogeneous interactions with the first ß-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the membrane integration of partially folded ß-barrels by a 'swing' mechanism.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Membrana Celular/química , Membrana Celular/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligação Proteica , Domínios Proteicos
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