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1.
Nat Commun ; 11(1): 3145, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561711

RESUMO

While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.

2.
Plant J ; 2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32365245

RESUMO

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

3.
Biochem J ; 477(9): 1631-1650, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32267468

RESUMO

Proton gradient regulation 5 (PGR5) is involved in the control of photosynthetic electron transfer, but its mechanistic role is not yet clear. Several models have been proposed to explain phenotypes such as a diminished steady-state proton motive force (pmf) and increased photodamage of photosystem I (PSI). Playing a regulatory role in cyclic electron flow (CEF) around PSI, PGR5 contributes indirectly to PSI protection by enhancing photosynthetic control, which is a pH-dependent down-regulation of electron transfer at the cytochrome b6f complex (b6f). Here, we re-evaluated the role of PGR5 in the green alga Chlamydomonas reinhardtii and conclude that pgr5 possesses a dysfunctional b6f. Our data indicate that the b6f low-potential chain redox activity likely operated in two distinct modes - via the canonical Q cycle during linear electron flow and via an alternative Q cycle during CEF, which allowed efficient oxidation of the low-potential chain in the WT b6f. A switch between the two Q cycle modes was dependent on PGR5 and relied on unknown stromal electron carrier(s), which were a general requirement for b6f activity. In CEF-favoring conditions, the electron transfer bottleneck in pgr5 was the b6f, in which insufficient low-potential chain redox tuning might account for the mutant pmf phenotype. By attributing a ferredoxin-plastoquinone reductase activity to the b6f and investigating a PGR5 cysteine mutant, a current model of CEF is challenged.

4.
Biochim Biophys Acta Bioenerg ; 1861(5-6): 148183, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32173384

RESUMO

Photosynthetic organisms are frequently exposed to excess light conditions and hence to photo-oxidative stress. To counteract photo-oxidative damage, land plants and most algae make use of non- photochemical quenching (NPQ) of excess light energy, in particular the rapidly inducible and relaxing qE-mechanism. In vascular plants, the constitutively active PsbS protein is the key regulator of qE. In the green algae C. reinhardtii, however, qE activation is only possible after initial high-light (HL) acclimation for several hours and requires the synthesis of LHCSR proteins which act as qE regulators. The precise function of PsbS, which is transiently expressed during HL acclimation in C. reinhardtii, is still unclear. Here, we investigated the impact of different PsbS amounts on HL acclimation characteristics of C. reinhardtii cells. We demonstrate that lower PsbS amounts negatively affect HL acclimation at different levels, including NPQ capacity, electron transport characteristics, antenna organization and morphological changes, resulting in an overall increased HL sensitivity and lower vitality of cells. Contrarily, higher PsbS amounts do not result in a higher NPQ capacity, but nevertheless provide higher fitness and tolerance towards HL stress. Strikingly, constitutively expressed PsbS protein was found to be degraded during HL acclimation. We propose that PsbS is transiently required during HL acclimation for the reorganization of thylakoid membranes and/or antenna proteins along with the activation of NPQ and adjustment of electron transfer characteristics, and that degradation of PsbS is essential in the fully HL acclimated state.

5.
J Biol Chem ; 295(1): 170-180, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31776187

RESUMO

Calcium (Ca2+) and redox signaling enable cells to quickly adapt to changing environments. The signaling protein calredoxin (CRX) from the green alga Chlamydomonas reinhardtii is a chloroplast-resident thioredoxin having Ca2+-dependent activity and harboring a unique combination of an EF-hand domain connected to a typical thioredoxin-fold. Using small-angle X-ray scattering (SAXS), FRET, and NMR techniques, we found that Ca2+-binding not only induces a conformational change in the EF-hand domain, but also in the thioredoxin domain, translating into the onset of thioredoxin redox activity. Functional analyses of CRX with genetically altered EF-hands revealed that EF-hand 4 is important for mediating the communication between the two domains. Moreover, we crystallized a variant (C174S) of the CRX target protein peroxiredoxin 1 (PRX1) at 2.4 Å resolution, modeled the interaction complex of the two proteins, and analyzed it by cross-linking and MS analyses, revealing that the interaction interface is located close to the active sites of both proteins. Our findings shed light on the Ca2+ binding-induced changes in CRX structure in solution at the level of the overall protein and individual domains and residues.

6.
Plant J ; 102(2): 230-245, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31777161

RESUMO

Nowadays, little information is available regarding the N-glycosylation pathway in the green microalga Chlamydomonas reinhardtii. Recent investigation demonstrated that C. reinhardtii synthesizes linear oligomannosides. Maturation of these oligomannosides results in N-glycans that are partially methylated and carry one or two xylose residues. One xylose residue was demonstrated to be a core ß(1,2)-xylose. Recently, N-glycoproteomic analysis performed on glycoproteins secreted by C. reinhardtii demonstrated that the xylosyltransferase A (XTA) was responsible for the addition of the core ß(1,2)-xylose. Furthermore, another xylosyltransferase candidate named XTB was suggested to be involved in the xylosylation in C. reinhardtii. In the present study, we focus especially on the characterization of the structures of the xylosylated N-glycans from C. reinhardtii taking advantage of insertional mutants of XTA and XTB, and of the XTA/XTB double-mutant. The combination of mass spectrometry approaches allowed us to identify the major N-glycan structures bearing one or two xylose residues. They confirm that XTA is responsible for the addition of the core ß(1,2)-xylose, whereas XTB is involved in the addition of the xylose residue onto the linear branch of the N-glycan as well as in the partial addition of the core ß(1,2)-xylose suggesting that this transferase exhibits a low substrate specificity. Analysis of the double-mutant suggests that an additional xylosyltransferase is involved in the xylosylation process in C. reinhardtii. Additional putative candidates have been identified in the C. reinhardtii genome. Altogether, these results pave the way for a better understanding of the C. reinhardtii N-glycosylation pathway.

7.
Anal Chem ; 91(20): 13096-13104, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525022

RESUMO

We introduce and compare two powerful new techniques for headspace gas analysis above bacterial batch cultures by spectroscopy, Raman spectroscopy enhanced in an optical cavity (CERS), and photoacoustic detection in a differential Helmholtz resonator (DHR). Both techniques are able to monitor O2 and CO2 and its isotopomers with excellent sensitivity and time resolution to characterize bacterial growth and metabolism. We discuss and show some of the shortcomings of more conventional optical density (OD) measurements if used on their own without more sophisticated complementary measurements. The spectroscopic measurements can clearly and unambiguously distinguish the main phases of bacterial growth in the two media studied, LB and M9. We demonstrate how 13C isotopic labeling of sugars combined with spectroscopic detection allows the study of bacterial mixed sugar metabolism to establish whether sugars are sequentially or simultaneously metabolized. For E. coli, we have characterized the shift from glucose to lactose metabolism without a classic diauxic lag phase. DHR and CERS are shown to be cost-effective and highly selective analytical tools in the biosciences and in biotechnology, complementing and superseding existing conventional techniques. They also provide new capabilities for mechanistic investigations and show a great deal of promise for use in stable isotope bioassays.

8.
Anal Bioanal Chem ; 411(17): 3777-3787, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31111181

RESUMO

Photoacoustic spectroscopy in a differential Helmholtz resonator has been employed with near-IR and red diode lasers for the detection of CO2, H2S and O2 in 1 bar of air/N2 and natural gas, in static and flow cell measurements. With the red distributed feedback (DFB) diode laser, O2 can be detected at 764.3 nm with a noise equivalent detection limit of 0.60 mbar (600 ppmv) in 1 bar of air (35-mW laser, 1-s integration), corresponding to a normalised absorption coefficient α = 2.2 × 10-8 cm-1 W s1/2. Within the tuning range of the near-IR DFB diode laser (6357-6378 cm-1), CO2 and H2S absorption features can be accessed, with a noise equivalent detection limit of 0.160 mbar (160 ppmv) CO2 in 1 bar N2 (30-mW laser, 1-s integration), corresponding to a normalised absorption coefficient α = 8.3 × 10-9 cm-1 W s1/2. Due to stronger absorptions, the noise equivalent detection limit of H2S in 1 bar N2 is 0.022 mbar (22 ppmv) at 1-s integration time. Similar detection limits apply to trace impurities in 1 bar natural gas. Detection limits scale linearly with laser power and with the square root of integration time. At 16-s total measurement time to obtain a spectrum, a noise equivalent detection limit of 40 ppmv CO2 is obtained after a spectral line fitting procedure, for example. Possible interferences due to weak water and methane absorptions have been discussed and shown to be either negligible or easy to correct. The setup has been used for simultaneous in situ monitoring of O2, CO2 and H2S in the cysteine metabolism of microbes (E. coli), and for the analysis of CO2 and H2S impurities in natural gas. Due to the inherent signal amplification and noise cancellation, photoacoustic spectroscopy in a differential Helmholtz resonator has a great potential for trace gas analysis, with possible applications including safety monitoring of toxic gases and applications in the biosciences and for natural gas analysis in petrochemistry. Graphical abstract.


Assuntos
Ar/análise , Dióxido de Carbono/análise , Sulfeto de Hidrogênio/análise , Gás Natural/análise , Oxigênio/análise , Técnicas Fotoacústicas/métodos , Análise Espectral/métodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Limite de Detecção
9.
Plant J ; 99(5): 877-894, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31033075

RESUMO

Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N-terminal LHCSR3 phosphorylation. Phosphorylated and nonphosphorylated LHCSR3 associated with PSII-LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N-terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII-LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX, and FNR. This finding indicated the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow, and the oxidative stress response.

10.
Photosynth Res ; 139(1-3): 499-508, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29582227

RESUMO

The evolution of photosynthesis from primitive photosynthetic bacteria to higher plants has been driven by the need to adapt to a wide range of environmental conditions. The red alga Cyanidioschyzon merolae is a primitive organism, which is capable of performing photosynthesis in extreme acidic and hot environments. The study of its photosynthetic machinery may provide new insight on the evolutionary path of photosynthesis and on light harvesting and its regulation in eukaryotes. With that aim, the structural and functional properties of the PSI complex were investigated by biochemical characterization, mass spectrometry, and X-ray crystallography. PSI was purified from cells grown at 25 and 42 °C, crystallized and its crystal structure was solved at 4 Å resolution. The structure of C. merolae reveals a core complex with a crescent-shaped structure, formed by antenna proteins. In addition, the structural model shows the position of PsaO and PsaM. PsaG and PsaH are present in plant complex and are missing from the C. merolae model as expected. This paper sheds new light onto the evolution of photosynthesis, which gives a strong indication for the chimerical properties of red algae PSI. The subunit composition of the PSI core from C. merolae and its associated light-harvesting antennae suggests that it is an evolutionary and functional intermediate between cyanobacteria and plants.


Assuntos
Cianobactérias/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Fotossíntese/fisiologia , Synechocystis/metabolismo
11.
New Phytol ; 221(4): 1935-1949, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30320882

RESUMO

RBOHF from Arabidopsis thaliana represents a multifunctional NADPH oxidase regulating biotic and abiotic stress tolerance, developmental processes and guard cell aperture. The molecular components and mechanisms determining RBOHF activity remain to be elucidated. Here we combined protein interaction studies, biochemical and genetic approaches, and pathway reconstitution analyses to identify and characterize proteins that confer RBOHF regulation and elucidated mechanisms that adjust RBOHF activity. While the Ca2+ sensor-activated kinases CIPK11 and CIPK26 constitute alternative paths for RBOHF activation, the combined activity of CIPKs and the kinase open stomata 1 (OST1) triggers complementary activation of this NADPH oxidase, which is efficiently counteracted through dephosphorylation by the phosphatase ABI1. Within RBOHF, several distinct phosphorylation sites (p-sites) in the N-terminus of RBOHF appear to contribute individually to activity regulation. These findings identify RBOHF as a convergence point targeted by a complex regulatory network of kinases and phosphatases. We propose that this allows for fine-tuning of plant reactive oxygen species (ROS) production by RBOHF in response to different stimuli and in diverse physiological processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , NADPH Oxidases/metabolismo , Arabidopsis/genética , Ativação Enzimática , Regulação da Expressão Gênica de Plantas , Células HEK293 , Humanos , Modelos Biológicos , Mutação/genética , Fenótipo , Fosforilação , Espécies Reativas de Oxigênio/metabolismo
12.
FEBS Lett ; 593(3): 339-351, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30556127

RESUMO

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2-type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8-ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca-1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA-induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Ácido Abscísico/genética , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Protoplastos/enzimologia , Tabaco/genética , Tabaco/metabolismo
13.
Proc Natl Acad Sci U S A ; 115(41): 10517-10522, 2018 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-30254175

RESUMO

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Complexo Citocromos b6f/química , Elétrons , Complexos de Proteínas Captadores de Luz/química , Complexos Multiproteicos/química , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Anaerobiose , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Complexo Citocromos b6f/metabolismo , Transporte de Elétrons , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Conformação Proteica
14.
J Biol Chem ; 293(45): 17559-17573, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30228184

RESUMO

The supramolecular organization of membrane proteins (MPs) is sensitive to environmental changes in photosynthetic organisms. Isolation of MP supercomplexes from the green algae Chlamydomonas reinhardtii, which are believed to contribute to cyclic electron flow (CEF) between the cytochrome b 6 f complex (Cyt-b 6 f) and photosystem I (PSI), proved difficult. We were unable to isolate a supercomplex containing both Cyt-b 6 f and PSI because in our hands, most of Cyt-b 6 f did not comigrate in sucrose density gradients, even upon using chemical cross-linkers or amphipol substitution of detergents. Assisted by independent affinity purification and MS approaches, we utilized disintegrating MP assemblies and demonstrated that the algae-specific CEF effector proteins PETO and ANR1 are bona fide Cyt-b 6 f interactors, with ANR1 requiring the presence of an additional, presently unknown, protein. We narrowed down the Cyt-b 6 f interface, where PETO is loosely attached to cytochrome f and to a stromal region of subunit IV, which also contains phosphorylation sites for the STT7 kinase.


Assuntos
Chlamydomonas reinhardtii/enzimologia , Complexo Citocromos b6f/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Chlamydomonas reinhardtii/genética , Complexo Citocromos b6f/genética , Complexo de Proteína do Fotossistema I/genética
15.
Plant Physiol ; 178(2): 583-595, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30126869

RESUMO

In plants, the photosystem I (PSI) core complex stably associates with its light-harvesting chlorophyll a/b complex I (LHCI) to form the PSI-LHCI supercomplex. The vascular plant PSI core complex associates with four distinct LHCI subunits, whereas that of the green alga Chlamydomonas reinhardtii binds nine distinct LHCI subunits (LHCA1-LHCA9). The stoichiometry and configuration of these LHCI subunits in the PSI-LHCI supercomplex of C. reinhardtii remain controversial. Here, we determined the stoichiometry of the nine distinct LHCI subunits relative to PSI subunits through uniform labeling of total proteins using 14C. We separated the nine LHCI polypeptides by three different sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Our data revealed that the PSI-LHCI supercomplex contains two LHCA1 proteins and one of each of the other eight LHCI subunits. Subsequently, we identified their cross-linked products by immunodetection and mass spectrometry to determine the configuration of the 10 LHCI subunits within the PSI-LHCI supercomplex. Furthermore, analyses of PSI-LHCI complexes isolated from ΔLHCA2 and ΔLHCA5 mutants and oligomeric LHCI from a PSI-deficient (ΔpsaA/B) mutant provided supporting evidence for the LHCI subunit configuration. In conclusion, eight LHCI subunits bind to the PSI core at the site of PSAF subunit in two layers: LHCA1-LHCA8-LHCA7-LHCA3 from PSAG to PSAK, in the inner layer, and LHCA1-LHCA4-LHCA6-LHCA5 in the outer layer. The other two LHCI subunits, LHCA2 and LHCA9, bind PSAB between PSAG and PSAH, PSAG-LHCA9-LHCA2-PSAH. Our study provides new insights into the LHCI configuration linked to the PSI core.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Estruturais , Complexo de Proteína do Fotossistema I/metabolismo , Chlamydomonas reinhardtii/genética , Clorofila/metabolismo , Clorofila A/metabolismo , Imunoquímica , Mutação , Complexo de Proteína do Fotossistema I/genética , Espectrometria de Massas em Tandem
16.
Redox Biol ; 18: 114-123, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30007887

RESUMO

Carbon monoxide (CO)-releasing molecules (CORMs), mostly metal carbonyl compounds, are extensively used as experimental tools to deliver CO, a biological 'gasotransmitter', in mammalian systems. CORMs are also explored as potential novel antimicrobial drugs, effectively and rapidly killing bacteria in vitro and in animal models, but are reportedly benign towards mammalian cells. Ru-carbonyl CORMs, exemplified by CORM-3 (Ru(CO)3Cl(glycinate)), exhibit the most potent antimicrobial effects against Escherichia coli. We demonstrate that CORM-3 releases little CO in buffers and cell culture media and that the active antimicrobial agent is Ru(II), which binds tightly to thiols. Thus, thiols and amino acids in complex growth media - such as histidine, methionine and oxidised glutathione, but most pertinently cysteine and reduced glutathione (GSH) - protect both bacterial and mammalian cells against CORM-3 by binding and sequestering Ru(II). No other amino acids exert significant protective effects. NMR reveals that CORM-3 binds cysteine and GSH in a 1:1 stoichiometry with dissociation constants, Kd, of about 5 µM, while histidine, GSSG and methionine are bound less tightly, with Kd values ranging between 800 and 9000 µM. There is a direct positive correlation between protection and amino acid affinity for CORM-3. Intracellular targets of CORM-3 in both bacterial and mammalian cells are therefore expected to include GSH, free Cys, His and Met residues and any molecules that contain these surface-exposed amino acids. These results necessitate a major reappraisal of the biological effects of CORM-3 and related CORMs.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Monóxido de Carbono/farmacologia , Escherichia coli/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Rutênio/farmacologia , Antibacterianos/química , Antineoplásicos/química , Monóxido de Carbono/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Neoplasias/tratamento farmacológico , Compostos Organometálicos/química , Rutênio/química
17.
BMC Genomics ; 19(1): 376, 2018 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-29783951

RESUMO

BACKGROUND: Regulatory adjustments to acute and chronic temperature changes are highly important for aquatic ectotherms because temperature affects their metabolic rate as well as the already low oxygen concentration in water, which can upset their energy balance. This also applies to severe changes in food supply. Thus, we studied on a molecular level (transcriptomics and/or proteomics) the immediate responses to heat stress and starvation and the acclimation to different temperatures in two clonal isolates of the model microcrustacean Daphnia pulex from more or less stressful environments, which showed a higher (clone M) or lower (clone G) tolerance to heat and starvation. RESULTS: The transcriptomic responses of clone G to acute heat stress (from 20 °C to 30 °C) and temperature acclimation (10 °C, 20 °C, and 24 °C) and the proteomic responses of both clones to acute heat, starvation, and heat-and-starvation stress comprised environment-specific and clone-specific elements. Acute stress (in particular heat stress) led to an early upregulation of stress genes and proteins (e.g., molecular chaperones) and a downregulation of metabolic genes and proteins (e.g., hydrolases). The transcriptomic responses to temperature acclimation differed clearly. They also varied depending on the temperature level. Acclimation to higher temperatures comprised an upregulation of metabolic genes and, in case of 24 °C acclimation, a downregulation of genes for translational processes and collagens. The proteomic responses of the clones M and G differed at any type of stress. Clone M showed markedly stronger and less stress-specific proteomic responses than clone G, which included the consistent expression of a specific heat shock protein (HSP60) and vitellogenin (VTG-SOD). CONCLUSIONS: The expression changes under acute stress can be interpreted as a switch from standard products of gene expression to stress-specific products. The expression changes under temperature acclimation probably served for an increase in energy intake (via digestion) and, if necessary, a decrease in energy expenditures (e.g, for translational processes). The stronger and less stress-specific proteomic responses of clone M indicate a lower degree of cell damage and an active preservation of the energy balance, which allowed adequate proteomic responses under stress, including the initiation of resting egg production (VTG-SOD expression) as an emergency reaction.


Assuntos
Daphnia/genética , Daphnia/fisiologia , Meio Ambiente , Perfilação da Expressão Gênica , Proteômica , Temperatura , Aclimatação/genética , Animais , Abastecimento de Alimentos , Resposta ao Choque Térmico/genética
18.
Photosynth Res ; 137(2): 281-293, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29594952

RESUMO

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 µmolH2 min-1 mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Ferredoxinas/metabolismo , Hidrogenase/metabolismo , Nitrito Redutases/metabolismo , Fotossíntese/fisiologia , Plastocianina/metabolismo , Aerobiose , Sequência de Aminoácidos , Anaerobiose , Ferredoxinas/genética , Regulação da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica , Hidrogenase/genética , Nitrito Redutases/genética , Oxigênio/metabolismo , Oxigênio/farmacologia
19.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 2): 86-91, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29400317

RESUMO

Peroxiredoxins (PRXs) are a group of antioxidant enzymes that are found in all organisms, including plants and green algae. The 2-Cys PRX from Chlamydomonas reinhardtii (CrPRX1) is a chloroplast-localized protein that is critical for clearing reactive oxygen species in chloroplasts. CrPRX1 is reduced by thioredoxins or calredoxin (CrCRX), a recently identified calcium-dependent redox protein. The molecular interaction between PRXs and thioredoxin/CrCRX is functionally important, but discussion has been limited owing to a lack of structural information on CrPRX1, especially regarding its oligomeric state. In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers.


Assuntos
Chlamydomonas reinhardtii/genética , Microscopia de Força Atômica/métodos , Peroxirredoxinas/química , Peroxirredoxinas/genética , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , Peroxirredoxinas/isolamento & purificação
20.
New Phytol ; 218(2): 414-431, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29332310

RESUMO

Content Summary 414 I. Introduction 415 II. Ca2+ importer and exporter in plants 415 III. The Ca2+ decoding toolkit in plants 415 IV. Mechanisms of Ca2+ signal decoding 417 V. Immediate Ca2+ signaling in the regulation of ion transport 418 VI. Ca2+ signal integration into long-term ABA responses 419 VII Integration of Ca2+ and hormone signaling through dynamic complex modulation of the CCaMK/CYCLOPS complex 420 VIII Ca2+ signaling in mitochondria and chloroplasts 422 IX A view beyond recent advances in Ca2+ imaging 423 X Modeling approaches in Ca2+ signaling 424 XI Conclusions: Ca2+ signaling a still young blooming field of plant research 424 Acknowledgements 425 ORCID 425 References 425 SUMMARY: Temporally and spatially defined changes in Ca2+ concentration in distinct compartments of cells represent a universal information code in plants. Recently, it has become evident that Ca2+ signals not only govern intracellular regulation but also appear to contribute to long distance or even organismic signal propagation and physiological response regulation. Ca2+ signals are shaped by an intimate interplay of channels and transporters, and during past years important contributing individual components have been identified and characterized. Ca2+ signals are translated by an elaborate toolkit of Ca2+ -binding proteins, many of which function as Ca2+ sensors, into defined downstream responses. Intriguing progress has been achieved in identifying specific modules that interconnect Ca2+ decoding proteins and protein kinases with downstream target effectors, and in characterizing molecular details of these processes. In this review, we reflect on recent major advances in our understanding of Ca2+ signaling and cover emerging concepts and existing open questions that should be informative also for scientists that are currently entering this field of ever-increasing breath and impact.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Transporte de Íons , Proteínas de Membrana Transportadoras/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Plantas/metabolismo
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