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1.
BMC Plant Biol ; 21(1): 376, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399701

RESUMO

BACKGROUND: Glycolytic pathway is common in all plant organs, especially in oxygen-deficient tissues. Phosphofructokinase (PFK) is a rate-limiting enzyme in the glycolytic pathway and catalyses the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Cassava (M. esculenta) root is a huge storage organ with low amount of oxygen. However, less is known about the functions of PFK from M. esculenta (MePFK). We conducted a systematic analysis of MePFK genes to explore the function of the MePFK gene family under hypoxic stress. RESULTS: We identified 13 MePFK genes and characterised their sequence structure. The phylogenetic tree divided the 13 genes into two groups: nine were MePFKs and four were pyrophosphate-fructose-6-phosphate phosphotransferase (MePFPs). We confirmed by green fluorescent protein fusion protein expression that MePFK03 and MePFPA1 were localised in the chloroplast and cytoplasm, respectively. The expression profiles of the 13 MePFKs detected by quantitative reverse transcription polymerase chain reaction revealed that MePFK02, MePFK03, MePFPA1, MePFPB1 displayed higher expression in leaves, root and flower. The expression of MePFK03, MePFPA1 and MePFPB1 in tuber root increased gradually with plant growth. We confirmed that hypoxia occurred in the cassava root, and the concentration of oxygen was sharply decreasing from the outside to the inside root. The expression of MePFK03, MePFPA1 and MePFPB1 decreased with the decrease in the oxygen concentration in cassava root. Waterlogging stress treatment showed that the transcript level of PPi-dependent MePFP and MeSuSy were up-regulated remarkably and PPi-dependent glycolysis bypass was promoted. CONCLUSION: A systematic survey of phylogenetic relation, molecular characterisation, chromosomal and subcellular localisation and cis-element prediction of MePFKs were performed in cassava. The expression profiles of MePFKs in different development stages, organs and under waterlogging stress showed that MePFPA1 plays an important role during the growth and development of cassava. Combined with the transcriptional level of MeSuSy, we found that pyrophosphate (PPi)-dependent glycolysis bypass was promoted when cassava was under waterlogging stress. The results would provide insights for further studying the function of MePFKs under hypoxic stress.


Assuntos
Genoma de Planta , Manihot/enzimologia , Manihot/genética , Fosfofrutoquinases/genética , Fosfofrutoquinases/metabolismo , Cloroplastos/enzimologia , Mapeamento Cromossômico , Cromossomos de Plantas , Sequência Conservada , Citoplasma/enzimologia , Éxons , Flores/enzimologia , Íntrons , Família Multigênica , Oxigênio/metabolismo , Filogenia , Folhas de Planta/enzimologia , Raízes de Plantas/enzimologia , Regiões Promotoras Genéticas , Estresse Fisiológico/genética , Transcriptoma
2.
Gene ; 800: 145836, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34280510

RESUMO

Skeletal muscle atrophy can result from a range of physiological conditions, including denervation, immobilization, hindlimb unweighting, and aging. To better characterize the molecular genetic events of atrophy, a microarray analysis revealed that FGGY carbohydrate kinase domain containing (Fggy) is expressed in skeletal muscle and is induced in response to denervation. Bioinformatic analysis of the Fggy gene locus revealed two validated isoforms with alternative transcription initiation sites that we have designated Fggy-L-552 and Fggy-S-387. Additionally, we cloned two novel alternative splice variants, designated Fggy-L-482 and Fggy-S-344, from cultured muscle cells suggesting that at least four Fggy splice variants are expressed in skeletal muscle. Quantitative RT-PCR was performed using RNA isolated from muscle cells and primers designed to distinguish the four alternative Fggy transcripts and found that the Fggy-L transcripts are more highly expressed during myoblast differentiation, while the Fggy-S transcripts show relatively stable expression in proliferating myoblasts and differentiated myotubes. Confocal fluorescent microscopy revealed that the Fggy-L variants appear to localize evenly throughout the cytoplasm, while the Fggy-S variants produce a more punctuate cytoplasmic localization pattern in proliferating muscle cells. Finally, ectopic expression of Fggy-L-552 and Fggy-S-387 resulted in inhibition of muscle cell differentiation and attenuation of the MAP kinase and Akt signaling pathways. The identification and characterization of novel genes such as Fggy helps to improve our understanding of the molecular and cellular events that lead to atrophy and may eventually result in the identification of new therapeutic targets for the treatment of muscle wasting.


Assuntos
Músculo Esquelético/enzimologia , Atrofia Muscular/genética , Fosfotransferases/genética , Fosfotransferases/metabolismo , Sítios de Splice de RNA , Animais , Diferenciação Celular/genética , Células Cultivadas , Citoplasma/enzimologia , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
3.
Plant J ; 106(3): 706-719, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33570751

RESUMO

Phosphorus is a crucial macronutrient for plant growth and development. The mechanisms for maintaining inorganic phosphate (Pi) homeostasis in rice are not well understood. The ubiquitin-conjugating enzyme variant protein OsUEV1B was previously found to interact with OsUbc13 and mediate lysine63-linked polyubiquitination. In the present study, we found OsUEV1B was specifically inhibited by Pi deficiency, and was localized in the nucleus and cytoplasm. Both osuev1b mutant and OsUEV1B-RNA interference (RNAi) lines displayed serious symptoms of toxicity due to Pi overaccumulation. Some Pi starvation inducible and phosphate transporter genes were upregulated in osuev1b mutant and OsUEV1B-RNAi plants in association with enhanced Pi acquisition, and representative Pi starvation responses, including stimulation of acid phosphatase activity and root hair growth, were also activated in the presence of sufficient Pi. A yeast two-hybrid screen revealed an interaction between OsUEV1B and OsVDAC1, which was confirmed by bimolecular fluorescence complementation and firefly split-luciferase complementation assays. OsVDAC1 encoded a voltage-dependent anion channel protein localized in the mitochondria, and OsUbc13 was shown to interact with OsVDAC1 via yeast two-hybrid and bimolecular fluorescence complementation assays. Under sufficient Pi conditions, similar to osuev1b, a mutation in OsVDAC1 resulted in significantly greater Pi concentrations in the roots and second leaves, improved acid phosphatase activity, and enhanced expression of the Pi starvation inducible and phosphate transporter genes compared with wild-type DongJin, whereas overexpression of OsVDAC1 had the opposite effects. OsUEV1B or OsVDAC1 knockout reduced the mitochondrial membrane potential and adenosine triphosphate levels. Moreover, overexpression of OsVDAC1 in osuev1b partially restored its high Pi concentration to a level between those of osuev1b and DongJin. Our results indicate that OsUEV1B is required for rice phosphate homeostasis.


Assuntos
Homeostase , Oryza/metabolismo , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/metabolismo , Citoplasma/enzimologia , Citoplasma/metabolismo , Oryza/enzimologia , Proteínas de Plantas/fisiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/metabolismo , Brotos de Planta/enzimologia , Brotos de Planta/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia
4.
Nucleic Acids Res ; 49(5): 2816-2834, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619562

RESUMO

GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases.


Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Animais , Domínio Catalítico , Citoplasma/enzimologia , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Modelos Moleculares , Mutação , Transporte Proteico , Proteínas de Ligação a RNA/metabolismo , Células Sf9
5.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33472976

RESUMO

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.


Assuntos
Proteínas de Bactérias/química , Glicoconjugados/química , Glicosiltransferases/química , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , Polissacarídeos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Citoplasma/enzimologia , Citoplasma/genética , Evolução Molecular , Expressão Gênica , Redes Reguladoras de Genes , Glicoconjugados/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Redes e Vias Metabólicas/genética , Modelos Moleculares , Periplasma/enzimologia , Periplasma/genética , Filogenia , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
6.
FEBS J ; 288(2): 566-581, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32359191

RESUMO

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.


Assuntos
Processamento Alternativo , Núcleo Celular/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/química , Sequência Conservada , Citoplasma/química , Citoplasma/enzimologia , Expressão Gênica , Células HeLa , Humanos , Cinética , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
7.
Biochim Biophys Acta Proteins Proteom ; 1869(2): 140575, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33242654

RESUMO

BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.


Assuntos
Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Redutases/genética , Metionina/metabolismo , Sequência de Aminoácidos/genética , Catálise , Citoplasma/enzimologia , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimologia , Metionina/química , Metionina/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/ultraestrutura , Oxirredução , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato
8.
Prostate ; 80(14): 1188-1202, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33258506

RESUMO

BACKGROUND: As the survival of castration-resistant prostate cancer (CRPC) remains poor, and the nuclear factor-κB (NF-κB) pathways play key roles in prostate cancer (PC) progression, several studies have focused on inhibiting the NF-κB pathway through generating inhibitory κB kinase subunit α (IKKα) small molecule inhibitors. However, the identification of prognostic markers able to discriminate which patients could benefit from IKKα inhibitors is urgently required. The present study investigated the prognostic value of IKKα, IKKα phosphorylated at serine 180 (p-IKKα S180) and threonine 23 (p-IKKα T23), and their relationship with the androgen receptor (AR) and Ki67 proliferation index to predict patient outcome. METHODS: A cohort of 115 patients with hormone-naïve PC (HNPC) and CRPC specimens available were used to assess tumor cell expression of proteins within both the cytoplasm and the nucleus by immunohistochemistry. The expression levels were dichotomized (low vs high) to determine the associations between IKKα, AR, Ki67, and patients'Isurvival. In addition, an analysis was performed to assess potential IKKα associations with clinicopathological and inflammatory features, and potential IKKα correlations with other cancer pathways essential for CRPC growth. RESULTS: High levels of cytoplasmic IKKα were associated with a higher cancer-specific survival in HNPC patients with low AR expression (hazards ratio [HR], 0.33; 95% confidence interval [CI] log-rank, 0.11-0.98; P = .04). Furthermore, nuclear IKKα (HR, 2.60; 95% CI, 1.27-5.33; P = .01) and cytoplasmic p-IKKα S180 (HR, 2.10; 95% CI, 1.17-3.76; P = .01) were associated with a lower time to death from recurrence in patients with CRPC. In addition, high IKKα expression was associated with high levels of T-cells (CD3+ P = .01 and CD8+ P = .03) in HNPC; however, under castration conditions, high IKKα expression was associated with high levels of CD68+ macrophages (P = .04), higher Gleason score (P = .01) and more prostate-specific antigen concentration (P = .03). Finally, we identified crosstalk between IKKα and members of the canonical NF-κB pathway in the nucleus of HNPC. Otherwise, IKKα phosphorylated by noncanonical NF-κB and Akt pathways correlated with members of the canonical NF-κB pathway in CRPC. CONCLUSION: The present study reports that patients with CRPC expressing high levels of nuclear IKKα or cytoplasmic p-IKKα S180, which associated with a lower time to death from recurrence, may benefit from IKKα inhibitors.


Assuntos
Quinase I-kappa B/metabolismo , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias da Próstata/enzimologia , Idoso , Biomarcadores Tumorais/metabolismo , Núcleo Celular/enzimologia , Estudos de Coortes , Citoplasma/enzimologia , Humanos , Quinase I-kappa B/imunologia , Imunidade Inata , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Prognóstico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/imunologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Taxa de Sobrevida
9.
mSphere ; 5(6)2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328349

RESUMO

In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 µM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.


Assuntos
Parede Celular/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Saccharomyces cerevisiae/metabolismo , Secreções Corporais/metabolismo , Permeabilidade da Membrana Celular , Citoplasma/enzimologia , Citometria de Fluxo , Gliceraldeído-3-Fosfato Desidrogenases/química , Saccharomyces cerevisiae/crescimento & desenvolvimento
10.
Science ; 370(6519)2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33214250

RESUMO

The photosynthetic apparatus of green sulfur bacteria (GSB) contains a peripheral antenna chlorosome, light-harvesting Fenna-Matthews-Olson proteins (FMO), and a reaction center (GsbRC). We used cryo-electron microscopy to determine a 2.7-angstrom structure of the FMO-GsbRC supercomplex from Chlorobaculum tepidum The GsbRC binds considerably fewer (bacterio)chlorophylls [(B)Chls] than other known type I RCs do, and the organization of (B)Chls is similar to that in photosystem II. Two BChl layers in GsbRC are not connected by Chls, as seen in other RCs, but associate with two carotenoid derivatives. Relatively long distances of 22 to 33 angstroms were observed between BChls of FMO and GsbRC, consistent with the inefficient energy transfer between these entities. The structure contains common features of both type I and type II RCs and provides insight into the evolution of photosynthetic RCs.


Assuntos
Proteínas de Bactérias/química , Chlorobi/enzimologia , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema II/química , Microscopia Crioeletrônica , Citoplasma/enzimologia , Transferência de Energia , Periplasma/enzimologia , Conformação Proteica
11.
J Mol Biol ; 432(23): 6108-6126, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33058874

RESUMO

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.


Assuntos
Dano ao DNA/genética , Fumarato Hidratase/genética , Processamento de Proteína Pós-Traducional/genética , Respiração/genética , Citoplasma/enzimologia , Citoplasma/genética , Reparo do DNA/genética , Fumarato Hidratase/química , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Fosforilação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Succínico/metabolismo , Ubiquitinação/genética
12.
Asian Pac J Cancer Prev ; 21(9): 2739-2750, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32986376

RESUMO

OBJECTIVE: In search for a unique natural combination of highly active biological components for treatment against colon cancer, we used aqueous extract of Ascidia, Styela plicata (ASCex), a marine invertebrate depending on its richness of high levels of biologically active components as indicated in our previous studies, against rat colon cancer, exploring its underlying mechanisms. METHODS: Rats chemically initiated for colon cancer were either non-treated or post-treated with highly saturated ASCex for 32 weeks after initiation, other groups of rats were administered ASCex without cancer initiation or served as normal controls. RESULTS: Rats treated with ASCex alone did not show any signs of non-favored health conditions. Treatment with ASCex after cancer initiation has significantly reduced the average incidences, multiplicities and volumes of colon tumors (adenomas and adenocarcinomas) as compared with the non-treated cancer group. ASCex has also significantly reduced the total numbers of aberrant crypt foci (ACF), surrogate biomarkers for colon cancer as compared with the non-treated cancer group. Moreover, anti-proliferative celluar nucular antigen (PCNA) immunohistochemical staining revealed that ASCex exerted significant antiproliferative characteristics in the carcinogen-treated colonic mucosa as compared with its corresponding control. Also, treatment with ASCex has markedly down-regulated the mRNA expression levels of Nuclear Factor-kappa B (NF-κB), a nuclear transcriptional activator as well as the mRNA expression of the cytoplasmic SOD1 gene which encodes Cu/Zn SOD, the first line defense against superoxide radicals. CONCLUSION: Collectively, ASCex could act as a potent chemotherapeutic drug against colon cancer, likely through the influence of its rich active metabolites which interfere with various biological pathways including inhibition of protein synthesis during cellular growth and marked induction of antioxidative capacity in the colonic mucosa. This role has been extensively discussed herein.


Assuntos
Carcinogênese/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Superóxido Dismutase-1/metabolismo , Urocordados/química , Animais , Azoximetano/toxicidade , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinógenos/toxicidade , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citoplasma/enzimologia , Masculino , NF-kappa B/genética , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase-1/genética
13.
Proc Natl Acad Sci U S A ; 117(38): 23565-23570, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900959

RESUMO

l-cysteine is the source of all bacterial sulfurous biomolecules. However, the cytoplasmic level of l-cysteine must be tightly regulated due to its propensity to reduce iron and drive damaging Fenton chemistry. It has been proposed that in Escherichia coli the component of cytochrome bd-I terminal oxidase, the CydDC complex, shuttles excessive l-cysteine from the cytoplasm to the periplasm, thereby maintaining redox homeostasis. Here, we provide evidence for an alternative function of CydDC by demonstrating that the cydD phenotype, unlike that of the bona fide l-cysteine exporter eamA, parallels that of the l-cystine importer tcyP. Chromosomal induction of eamA, but not of cydDC, from a strong pLtetO-1 promoter (Ptet) leads to the increased level of extracellular l-cysteine, whereas induction of cydDC or tcyP causes the accumulation of cytoplasmic l-cysteine. Congruently, inactivation of cydD renders cells resistant to hydrogen peroxide and to aminoglycoside antibiotics. In contrast, induction of cydDC sensitizes cells to oxidative stress and aminoglycosides, which can be suppressed by eamA overexpression. Furthermore, inactivation of the ferric uptake regulator (fur) in Ptet-cydDC or Ptet-tcyP cells results in dramatic loss of survival, whereas catalase (katG) overexpression suppresses the hypersensitivity of both strains to H2O2 These results establish CydDC as a reducer of cytoplasmic cystine, as opposed to an l-cysteine exporter, and further elucidate a link between oxidative stress, antibiotic resistance, and sulfur metabolism.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cisteína/metabolismo , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/metabolismo , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Aminoglicosídeos/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Grupo dos Citocromos b/genética , Citoplasma/enzimologia , Citoplasma/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Proteínas de Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , NADH NADPH Oxirredutases/genética , Estresse Oxidativo/genética , Oxirredutases/genética , Periplasma/metabolismo
14.
Nat Commun ; 11(1): 3060, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561735

RESUMO

The MLKL pseudokinase is the terminal effector in the necroptosis cell death pathway. Phosphorylation by its upstream regulator, RIPK3, triggers MLKL's conversion from a dormant cytoplasmic protein into oligomers that translocate to, and permeabilize, the plasma membrane to kill cells. The precise mechanisms underlying these processes are incompletely understood, and were proposed to differ between mouse and human cells. Here, we examine the divergence of activation mechanisms among nine vertebrate MLKL orthologues, revealing remarkable specificity of mouse and human RIPK3 for MLKL orthologues. Pig MLKL can restore necroptotic signaling in human cells; while horse and pig, but not rat, MLKL can reconstitute the mouse pathway. This selectivity can be rationalized from the distinct conformations observed in the crystal structures of horse and rat MLKL pseudokinase domains. These studies identify important differences in necroptotic signaling between species, and suggest that, more broadly, divergent regulatory mechanisms may exist among orthologous pseudoenzymes.


Assuntos
Proteínas Quinases/química , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Animais , Galinhas , Cristalografia por Raios X , Citoplasma/enzimologia , Células HEK293 , Cavalos , Humanos , Camundongos , Necroptose , Necrose/metabolismo , Fosforilação , Conformação Proteica , Ratos , Transdução de Sinais , Smegmamorpha , Suínos , Células U937 , Xenopus
15.
Int J Mol Sci ; 21(12)2020 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545723

RESUMO

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl cis/trans isomerases (PPIs). The Escherichia coli cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6ppi strains could be constructed only on minimal medium in the temperature range of 30-37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the ppiC gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC's PPIase activity was shown to be required for interaction with AhpC.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Calorimetria , Citoplasma/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Mutação , Peptidilprolil Isomerase/genética , Dobramento de Proteína , Especificidade por Substrato
16.
Cell Physiol Biochem ; 54(3): 474-492, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32392404

RESUMO

BACKGROUND/AIMS: The subcellular localization of ERK1 and ERK2 (ERKs) in cells, which is important for proper signaling, may be regulated through protein-protein interactions. However, the proteins involved and the way they are regulated to affect localization is not entirely understood. METHODS: In order to identify the interacting proteins upon varying conditions, we used co-immunoprecipitation of ERK, active ERK and its binding CRS mutant. In addition, we examined the effect of intracellular calcium on the binding using calcium chelators and ionophores, analyzing the binding using silver stain, mass spectrometry and immunoblotting. The effect of calcium on ERK localization was examined using immunofluorescent staining and Western blotting. RESULTS: We found that inactive ERK2 interacts with a large number of proteins through its CRS/CD domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERKs' interactions influenced also the localization of ERKs, as calcium chelators enhanced nuclear translocation, while elevated calcium levels prevented it. This effect of calcium was also apparent upon the physiological lysophosphatidic acid stimulation, where ERKs translocation was delayed compared to that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERKs' translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins such as NUP153. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. CONCLUSION: Upon stimulation, ERKs are phosphorylated and released from their cytoplasmic anchors to allow shuttling into the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and therefore also their spatiotemporal regulation. Thus, calcium regulates ERKs localization, which is important for the compartmentalization of ERKs with their proper substrates, and thereby their signaling specificity.


Assuntos
Cálcio/metabolismo , Núcleo Celular/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/enzimologia , Citoplasma/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/genética , Sistema de Sinalização das MAP Quinases , Espectrometria de Massas , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Ratos
17.
Am J Physiol Regul Integr Comp Physiol ; 318(5): R1004-R1013, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32292063

RESUMO

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.


Assuntos
Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/toxicidade , Animais , Morte Celular , Linhagem Celular Tumoral , Citoplasma/enzimologia , Citoproteção , Inibidores Enzimáticos/farmacologia , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/patologia , Masculino , Peroxirredoxinas/antagonistas & inibidores , Peroxirredoxinas/genética , Quinoxalinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismo
18.
Protein Expr Purif ; 171: 105625, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32173567

RESUMO

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol. It also has the intrinsic ability to in situ detoxify aldehydes derived from lignocellulosic hydrolysis. However, the aldehyde reductases that catalyze this bioconversion in C. tropicalis remain unknown. Herein, we found that the uncharacterized open reading frame (ORF), CTRG_02797, from C. tropicalis encodes a novel and broad substrate-specificity aldehyde reductase that reduces at least seven aldehydes. This enzyme strictly depended on NADH rather than NADPH as the co-factor for catalyzing the reduction reaction. Its highest affinity (Km), maximum velocity (Vmax), catalytic rate constant (Kcat), and catalytic efficiency (Kcat/Km) were observed when reducing acetaldehyde (AA) and its enzyme activity was influenced by different concentrations of salts, metal ions, and chemical protective additives. Protein localization assay demonstrated that Ctrg_02797p was localized in the cytoplasm in C. tropicalis cells, which ensures an effective enzymatic reaction. Finally, Ctrg_02797p was grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily of the medium-chain dehydrogenase/reductase family. This research provides guidelines for exploring more uncharacterized genes with reduction activity for detoxifying aldehydes.


Assuntos
Aldeído Redutase/metabolismo , Candida tropicalis/enzimologia , Citoplasma/enzimologia , Proteínas Fúngicas/metabolismo , NADP/metabolismo , Fases de Leitura Aberta , Aldeído Redutase/genética , Candida tropicalis/genética , Citoplasma/genética , Proteínas Fúngicas/genética , NADP/genética
19.
Nucleic Acids Res ; 48(8): 3999-4012, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32201888

RESUMO

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.


Assuntos
Adenosina Desaminase/metabolismo , Neurônios/enzimologia , Edição de RNA , Adenosina/metabolismo , Animais , Axônios/enzimologia , Citoplasma/enzimologia , Decapodiformes/enzimologia , Células HEK293 , Humanos , Inosina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sinapses/enzimologia
20.
Nucleic Acids Res ; 48(7): 3922-3934, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32055835

RESUMO

In Escherichia coli, the endoribonuclease E (RNase E) can recruit several other ribonucleases and regulatory proteins via its noncatalytic domain to form an RNA degradosome that controls cellular RNA turnover. Similar RNA degradation complexes have been found in other bacteria; however, their compositions are varied among different bacterial species. In cyanobacteria, only the exoribonuclease PNPase was shown to bind to the noncatalytic domain of RNase E. Here, we showed that Alr1240, a member of the RNB family of exoribonucleases, could be co-isolated with RNase E from the lysate of the cyanobacterium Anabaena PCC 7120. Enzymatic analysis revealed that Alr1240 is an exoribonuclease II (RNase II), as it only degrades non-structured single-stranded RNA substrates. In contrast to known RNase E-interacting ribonucleases, which bind to the noncatalytic domain of RNase E, the Anabaena RNase II was shown to associate with the catalytic domain of RNase E. Using a strain in which RNase E and RNase II were tagged in situ with GFP and BFP, respectively, we showed that RNase E and RNase II form a compact complex in vivo by a fluorescence resonance energy transfer (FRET) assay. RNase E activity on several synthetic substrates was boosted in the presence of RNase II, suggesting that the activity of RNase E could be regulated by RNase II-RNase E interaction. To our knowledge, Anabaena RNase II is an unusual ribonuclease that interacts with the catalytic domain of RNase E, and it may represent a new type of RNA degradosome and a novel mechanism for regulating the activity of the RNA degradosome. As Anabaena RNase E interacts with RNase II and PNPase via different regions, it is very likely that the three ribonucleases form a large complex and cooperatively regulate RNA metabolism in the cell.


Assuntos
Anabaena/enzimologia , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Sítios de Ligação , Domínio Catalítico , Citoplasma/enzimologia , Endorribonucleases/química , Exorribonucleases/química
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