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1.
Adv Exp Med Biol ; 1274: 5-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894505

RESUMO

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , Aciltransferases/antagonistas & inibidores , Membrana Celular/metabolismo , Desenvolvimento de Medicamentos , Glicerofosfolipídeos/biossíntese , Glicerofosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Animais , Membrana Celular/química , Membrana Celular/enzimologia , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos
2.
Exp Parasitol ; 217: 107962, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763249

RESUMO

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.


Assuntos
FMN Redutase/metabolismo , Ferro/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Membrana Celular/enzimologia , Doença de Chagas/parasitologia , Colorimetria , FMN Redutase/análise , FMN Redutase/química , Imunofluorescência , Humanos , Filogenia , Distribuição de Poisson , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Trypanosoma cruzi/classificação , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Regulação para Cima
3.
Exp Parasitol ; 217: 107948, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32698076

RESUMO

Immunomodulation is an emerging concept to combat infection in recent years. Immunomodulators like arabinosylated-lipoarabinomannan (Ara-LAM) and glycyrrhizic-acid (GA) possess anti-leishmanial property, whereas sodium-antimony-gluconate (SAG) is still considered as the first choice for chemotherapy against leishmaniasis. During infection, invasion of Leishmania donovani needs the potential requirement of Ca2+, which is further responsible for apoptosis in intracellular amastigotes. However, suppression of elevated intracellular calcium by the activation of plasma-membrane-calcium-ATPase (PMCA4) facilitates survival of L. donovani in the host. In the present study, SAG, Ara-LAM, and GA were found to evoke significant increase in intracellular Ca2+ in L. donovani infected macrophages by inhibiting PMCA4. Moreover, PMCA4 inhibition by TFP or PMCA4 siRNA elevated the level of PKCß, whereas calcium-independent upregulation of PKCζ remained unchanged in infected macrophages. Furthermore, application of immunomodulators in infected macrophages resulted in down-regulation of PKCζ, conversion of anti-inflammatory to pro-inflammatory cytokines and inhibition of PMCA4. Plasma membrane-associated ceramide which is known to be elevated during leishmaniasis, triggered upregulation of PMCA4 via PKCζ activation. Interestingly, immunomodulators attenuated ceramide generation, which resulted into reduced PKCζ activation leading to the decreased PMCA expression in infected macrophages. Therefore, our study elucidated the efficacy of SAG, Ara-LAM, and GA in the reduction of parasite burden in macrophages by suppressing PMCA activation through inhibition of ceramide mediated upregulation of PKCζ.


Assuntos
Antiprotozoários/uso terapêutico , ATPases Transportadoras de Cálcio/sangue , Membrana Celular/enzimologia , Fatores Imunológicos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Animais , Gluconato de Antimônio e Sódio/farmacologia , Gluconato de Antimônio e Sódio/uso terapêutico , Antiprotozoários/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Ceramidas/metabolismo , Meios de Cultura Livres de Soro , Densitometria , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Ácido Glicirrízico/farmacologia , Ácido Glicirrízico/uso terapêutico , Imipramina/farmacologia , Immunoblotting , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/uso terapêutico , Macrófagos/fisiologia , Camundongos , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Transfecção
4.
Am J Physiol Cell Physiol ; 319(2): C250-C257, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579474

RESUMO

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca2+). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.


Assuntos
Eritrócitos/metabolismo , Canais Iônicos/genética , Mecanotransdução Celular/genética , Óxido Nítrico Sintase/genética , Cálcio/metabolismo , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/genética , Eritrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Canais Iônicos/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(26): 15006-15017, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554497

RESUMO

Cytochrome bo 3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo 3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.


Assuntos
Membrana Celular/enzimologia , Grupo dos Citocromos b/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Membrana Celular/química , Membrana Celular/genética , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fosfatidilcolinas/metabolismo , Polímeros/química , Prótons
6.
Artigo em Inglês | MEDLINE | ID: mdl-32348180

RESUMO

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.


Assuntos
Calpaína/deficiência , Membrana Celular/enzimologia , Músculo Esquelético/enzimologia , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Distrofia Muscular Animal/enzimologia , Animais , Proteínas de Bactérias/farmacologia , Sinalização do Cálcio , Calpaína/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Modelos Animais de Doenças , Disferlina/deficiência , Disferlina/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Saponinas/farmacologia , Índice de Gravidade de Doença , Estreptolisinas/farmacologia
7.
Plant Physiol Biochem ; 151: 429-437, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32289636

RESUMO

Potassium (K+) has been reported to alleviate ammonium (NH4+) toxicity in rice through some underlying mechanisms, but it still not clear. In addition, K+ is an important cation for activation of plasma membrane (PM) H+-ATPase activity. Here, we hypothesized that K+ alleviated NH4+ toxicity by mediating PM H+-ATPase function in rice root. In this study, rice plants were cultivated in hydroponic solution with various concentrations of K+ and NH4+. By concurrently supplying K+ with NH4+ or re-supplying K+ after NH4+ toxicity, we found that high K+ concentration reduced the NH4+ uptake rate, enhanced the H+ extrusion rate by the roots, and alleviated rice NH4+ toxicity. The gene expression levels of PM H+-ATPase members (OsA1, 3, 7, 8, and 9) were upregulated by application of increasing concentrations of K+ under NH4+ toxicity. The PM H+-ATPase activity and protein expression in rice roots were also enhanced. Furthermore, the enhancement of PM H+-ATPase activity by a specific stimulator (fusicoccin) rescued rice seedlings from NH4+ toxicity. Taken together, these results indicate that K+ can alleviate NH4+ toxicity, possibly by activating PM H+-ATPase to extrude more H+ and inhibit NH4+ uptake by root. Our results may enhance understanding of the strategy of applying K+ fertilizer to mitigate crop NH4+ toxicity in agriculture.


Assuntos
Compostos de Amônio , Oryza , Potássio , ATPases Translocadoras de Prótons , Compostos de Amônio/metabolismo , Compostos de Amônio/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/enzimologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Potássio/farmacologia , ATPases Translocadoras de Prótons/metabolismo
8.
Biochim Biophys Acta Bioenerg ; 1861(7): 148189, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194063

RESUMO

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F1 complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for FOF1 from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF1, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F1 with the membrane-embedded FO complex modulate inhibition by the εCTD. Here, we study membranes containing EcFOF1 with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).


Assuntos
Difosfato de Adenosina/farmacologia , Membrana Celular/enzimologia , Escherichia coli/enzimologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Dimetilaminas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hidrólise , Domínios Proteicos , Ácido Selenioso/farmacologia , Solubilidade
9.
Science ; 367(6483): 1240-1246, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165585

RESUMO

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a Legionella pneumophila effector protein. Cryo-electron microscopy allowed the construction of an atomic model, defining the enzyme's ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions.


Assuntos
Biomarcadores/química , Encéfalo/enzimologia , Receptores de Superfície Celular/química , ATPases Vacuolares Próton-Translocadoras/química , Animais , Proteínas de Bactérias/química , Biocatálise , Membrana Celular/enzimologia , Microscopia Crioeletrônica , Modelos Químicos , Domínios Proteicos , Ratos , Sistema Renina-Angiotensina , Via de Sinalização Wnt
10.
Microb Cell Fact ; 19(1): 52, 2020 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111210

RESUMO

BACKGROUND: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.


Assuntos
Bacillus subtilis/enzimologia , Membrana Celular/enzimologia , Peptídeo Hidrolases/biossíntese , Via Secretória , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptídeo Hidrolases/genética , Transporte Proteico , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , alfa-Amilases/genética
11.
Biochemistry (Mosc) ; 85(Suppl 1): S177-S195, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32087059

RESUMO

Enzymes of the phospholipase superfamily are involved in lipid metabolism, as well as regulation of membrane composition, cell signaling, and inflammation. This review provides an insight into the structure, functional properties, and biotechnological application of phospholipase A2 and phospholipases in general.


Assuntos
Biotecnologia , Indústria Alimentícia , Inibidores de Fosfolipase A2/uso terapêutico , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Animais , Membrana Celular/enzimologia , Expressão Gênica , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Isoenzimas , Metabolismo dos Lipídeos/fisiologia , Fosfolipases A2/classificação , Fosfolipases A2/genética , Estrutura Secundária de Proteína , Transdução de Sinais/fisiologia
12.
Int J Mol Sci ; 21(3)2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-32013007

RESUMO

Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida parapsilosis/crescimento & desenvolvimento , Anidrases Carbônicas/metabolismo , Técnicas de Cultura Celular por Lotes , Candida albicans/enzimologia , Candida parapsilosis/enzimologia , Membrana Celular/enzimologia , Parede Celular/enzimologia , Citosol/enzimologia , Proteínas Fúngicas/metabolismo , Espectrometria de Massas , Microscopia Eletrônica , Mitocôndrias/enzimologia
13.
Biochim Biophys Acta Biomembr ; 1862(4): 183193, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945321

RESUMO

Intramembrane proteases (IMPs) are proteolytic enzymes embedded in the lipid bilayer, where they cleave transmembrane substrates. The importance of IMPs relies on their role in a wide variety of cellular processes and diseases. In order to study the activity and function of IMPs, their purified form is often desired. The production of pure and active IMPs has proven to be a challenging task. This process unavoidably requires the use of solubilizing agents that will, to some extent, alter the native environment of these proteases. In this review we present the current solubilization and reconstitution techniques that have been applied to IMPs. In addition, we describe how these techniques had an influence on the activity and structural studies of IMPs, focusing on rhomboid proteases and γ-secretase.


Assuntos
Secretases da Proteína Precursora do Amiloide/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Peptídeo Hidrolases/isolamento & purificação , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Membrana Celular/química , Membrana Celular/enzimologia , Membrana Celular/genética , Microambiente Celular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-31913699

RESUMO

AMP-activated protein kinase (AMPK) activation promotes early stages of epithelial junction assembly. AMPK activation in MDCK renal epithelial cells facilitates localization of the junction-associated proteins aPKCζ and Par3 to the plasma membrane and promotes conversion of Cdc42, a key regulator of epithelial polarization and junction assembly, to its active GTP bound state. Furthermore, Par3 is an important regulator of AMPK-mediated aPKCζ localization. Both aPKCζ and Par3 serve as intermediates in AMPK-mediated junction assembly, with inhibition of aPKCζ activity or Par3 knockdown disrupting AMPK's ability to facilitate zonula occludens (ZO-1) localization. AMPK phosphorylates the adherens junction protein afadin and regulates its interaction with the tight-junction protein zonula occludens-1. Afadin is phosphorylated at two critical sites, S228 (residing within an aPKCζ consensus site) and S1102 (residing within an AMPK consensus site), that are differentially regulated during junction assembly and that exert different effects on the process. Expression of phospho-defective mutants (S228A and S1102A) perturbed ZO-1 localization to the plasma membrane during AMPK-induced junction assembly. Expression of S228A increased the ZO-1/afadin interaction, while S1102A reduced this interaction during extracellular calcium-induced junction assembly. Inhibition of aPKCζ activity also increased the ZO-1/afadin interaction. Taken together, these data suggest that aPKCζ phosphorylation of afadin terminates the ZO-1/afadin interaction and thus permits the later stages of junction assembly.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Membrana Celular/enzimologia , Junções Íntimas/enzimologia , Animais , Membrana Celular/química , Cães , Células Madin Darby de Rim Canino , Camundongos , Fosforilação/fisiologia , Proteína Quinase C/metabolismo , Junções Íntimas/química , Proteína da Zônula de Oclusão-1/metabolismo
15.
Photochem Photobiol Sci ; 19(1): 88-98, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31904040

RESUMO

Stomatal pores, which are surrounded by pairs of guard cells in the plant epidermis, regulate gas exchange between plants and the atmosphere, thereby controlling photosynthesis and transpiration. Blue light works as a signal to guard cells, to induce intracellular signaling and open stomata. Blue light receptor phototropins (phots) are activated by blue light; phot-mediated signals promote plasma membrane (PM) H+-ATPase activity via C-terminal Thr phosphorylation, serving as the driving force for stomatal opening in guard cells. However, the details of this signaling process are not fully understood. In this study, through an in vitro screening of phot-interacting protein kinases, we obtained the CBC1 and CBC2 that had been reported as signal transducers in stomatal opening. Promoter activities of CBC1 and CBC2 indicated that both genes were expressed in guard cells. Single and double knockout mutants of CBC1 and CBC2 showed no lesions in the context of phot-mediated phototropism, chloroplast movement, or leaf flattening. In contrast, the cbc1cbc2 double mutant showed larger stomatal opening under both dark and blue light conditions. Interestingly, the level of phosphorylation of C-terminal Thr of PM H+-ATPase was higher in double mutant guard cells. The larger stomatal openings of the double mutant were effectively suppressed by the phytohormone abscisic acid (ABA). CBC1 and CBC2 interacted with BLUS1 and PM H+-ATPase in vitro. From these results, we conclude that CBC1 and CBC2 act as negative regulators of stomatal opening, probably via inhibition of PM H+-ATPase activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/enzimologia , Estômatos de Plantas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Fosforilação
16.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31999306

RESUMO

Phosphatidic acid (PA) is both a central phospholipid biosynthetic intermediate and a multifunctional lipid second messenger produced at several discrete subcellular locations. Organelle-specific PA pools are believed to play distinct physiological roles, but tools with high spatiotemporal control are lacking for unraveling these pleiotropic functions. Here, we present an approach to precisely generate PA on demand on specific organelle membranes. We exploited a microbial phospholipase D (PLD), which produces PA by phosphatidylcholine hydrolysis, and the CRY2-CIBN light-mediated heterodimerization system to create an optogenetic PLD (optoPLD). Directed evolution of PLD using yeast membrane display and IMPACT, a chemoenzymatic method for visualizing cellular PLD activity, yielded a panel of optoPLDs whose range of catalytic activities enables mimicry of endogenous, physiological PLD signaling. Finally, we applied optoPLD to elucidate that plasma membrane, but not intracellular, pools of PA can attenuate the oncogenic Hippo signaling pathway. OptoPLD represents a powerful and precise approach for revealing spatiotemporally defined physiological functions of PA.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Membrana Celular/enzimologia , Optogenética , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipase D/metabolismo , Engenharia de Proteínas , Sistemas do Segundo Mensageiro , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Bactérias/genética , Células HEK293 , Humanos , Hidrólise , Membranas Intracelulares/enzimologia , Fosfolipase D/genética , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por Substrato , Fatores de Tempo , Fatores de Transcrição/metabolismo
17.
Biochim Biophys Acta Bioenerg ; 1861(2): 148132, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816290

RESUMO

Sulfide:quinone oxidoreductase (SQR) is a monotopic membrane flavoprotein present in all domains of life, with multiple roles including sulfide detoxification, homeostasis and energy generation by providing electrons to respiratory or photosynthetic electron transport chains. A type III SQR from the hyperthermophilic archeon Caldivirga maquilingensis has been previously characterized, and its C-terminal amphipathic helices were demonstrated to be responsible for membrane binding. Here, the oligomeric state of this protein was experimentally evaluated by size exclusion chromatography, native gels and crosslinking, and found to be a monomer-dimer-trimer equilibrium. Remarkably, mutant and truncated variants unable to bind to the membrane are able to maintain their oligomeric association. Thus, unlike other related monotopic membrane proteins, the region involved in membrane binding does not influence oligomerization. Furthermore, by studying heterodimers between the WT and mutants, it was concluded that membrane binding requires an oligomer with at least two copies of the protein with intact C-terminal amphipathic helices. A structural homology model of the C. maquilingensis SQR was used to define the flavin- and quinone-binding sites. CmGly12, CmGly16, CmAla77 and CmPro44 were determined to be important for flavin binding. Unexpectedly, CmGly299 is only important for quinone reduction despite its proximity to bound FAD. CmPhe337 and CmPhe362 are also important for quinone binding apparently by direct interaction with the quinone ring, whereas CmLys359, postulated to hydrogen bond to the quinone, seems to have a more structural role. The results presented differentiate the Type III CmSQR from some of its counterparts classified as Type I, II and V.


Assuntos
Proteínas Arqueais/química , Membrana Celular/enzimologia , NAD(P)H Desidrogenase (Quinona)/química , Multimerização Proteica , Thermoproteaceae/enzimologia , Proteínas Arqueais/metabolismo , Sítios de Ligação , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estrutura Secundária de Proteína
18.
Proc Natl Acad Sci U S A ; 117(2): 1167-1173, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879356

RESUMO

Chemiosmosis and substrate-level phosphorylation are the 2 mechanisms employed to form the biological energy currency adenosine triphosphate (ATP). During chemiosmosis, a transmembrane electrochemical ion gradient is harnessed by a rotary ATP synthase to phosphorylate adenosine diphosphate to ATP. In microorganisms, this ion gradient is usually composed of [Formula: see text], but it can also be composed of Na+ Here, we show that the strictly anaerobic rumen bacterium Pseudobutyrivibrio ruminis possesses 2 ATP synthases and 2 distinct respiratory enzymes, the ferredoxin:[Formula: see text] oxidoreductase (Rnf complex) and the energy-converting hydrogenase (Ech complex). In silico analyses revealed that 1 ATP synthase is [Formula: see text]-dependent and the other Na+-dependent, which was validated by biochemical analyses. Rnf and Ech activity was also biochemically identified and investigated in membranes of P. ruminis Furthermore, the physiology of the rumen bacterium and the role of the energy-conserving systems was investigated in dependence of 2 different catabolic pathways (the Embden-Meyerhof-Parnas or the pentose-phosphate pathway) and in dependence of Na+ availability. Growth of P. ruminis was greatly stimulated by Na+, and a combination of physiological, biochemical, and transcriptional analyses revealed the role of the energy conserving systems in P. ruminis under different metabolic scenarios. These data demonstrate the use of a 2-component ion circuit for [Formula: see text] bioenergetics and a 2nd 2-component ion circuit for Na+ bioenergetics in a strictly anaerobic rumen bacterium. In silico analyses infer that these 2 circuits are prevalent in a number of other strictly anaerobic microorganisms.


Assuntos
Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Clostridiales/metabolismo , Metabolismo Energético/fisiologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Clostridiales/enzimologia , Clostridiales/genética , Clostridiales/crescimento & desenvolvimento , Metabolismo Energético/genética , Ferredoxinas/metabolismo , Hidrogenase/metabolismo , Transporte de Íons , Oxirredução , Oxirredutases/metabolismo , Sódio/metabolismo
19.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
20.
Nature ; 576(7787): 477-481, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31827278

RESUMO

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis1. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells.


Assuntos
Membrana Celular/enzimologia , Proteína Oncogênica p21(ras)/metabolismo , Pinocitose , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Bicarbonatos/metabolismo , Carcinogênese , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Colesterol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Simportadores de Sódio-Bicarbonato/metabolismo
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