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1.
Plant Physiol ; 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34618078

RESUMO

Sulfur deficiency-induced proteins SDI1 and SDI2 play a fundamental role in sulfur homeostasis under sulfate-deprived conditions (-S) by downregulating glucosinolates. Here, we identified that besides glucosinolate regulation under -S, SDI1 downregulates another sulfur pool, the S-rich 2S seed storage proteins in Arabidopsis (Arabidopsis thaliana) seeds. We identified that MYB28 directly regulates 2S seed storage proteins by binding to the At2S4 promoter. We also showed that SDI1 downregulates 2S seed storage proteins by forming a ternary protein complex with MYB28 and MYC2, another transcription factor involved in the regulation of seed storage proteins. These findings have significant implications for the understanding of plant responses to sulfur deficiency.

2.
Comput Struct Biotechnol J ; 19: 5117-5125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34589187

RESUMO

Co-fractionation mass spectrometry (CF-MS)-based approaches enable cell-wide identification of protein-protein and protein-metabolite complexes present in the cellular lysate. CF-MS combines biochemical separation of molecular complexes with an untargeted mass-spectrometry-based proteomics and/or metabolomics analysis of the obtained fractions, and is used to delineate putative interactors. CF-MS data are a treasure trove for biological discovery. To facilitate analysis and visualization of original or publically available CF-MS datasets, we designed PROMISed, a user-friendly tool available online via https://myshiny.mpimp-golm.mpg.de/PDP1/ or as a repository via https://github.com/DennisSchlossarek/PROMISed. Specifically, starting with raw fractionation profiles, PROMISed (i) contains activities for data pre-processing and normalization, (ii) deconvolutes complex fractionation profiles into single, distinct peaks, (iii) identifies co-eluting protein-protein or protein-metabolite pairs using user-defined correlation methods, and (iv) performs co-fractionation network analysis. Given multiple CF-MS datasets, for instance representing different environmental condition, PROMISed allows to select for proteins and metabolites that differ in their elution profile, which may indicate change in the interaction status. But it also enables the identification of protein-protein and protein-metabolite pairs that co-elute together across multiple datasets. PROMISed enables users to (i) easily adjust parameters at each step of the analysis, (ii) download partial and final results, and (iii) select among different data-visualization options. PROMISed renders CF-MS data accessible to a broad scientific audience, allowing users with no computational or statistical background to look for novel protein-protein and protein-metabolite complexes for further experimental validation.

3.
Pharmaceuticals (Basel) ; 14(8)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34451801

RESUMO

TrkB is a tyrosine kinase receptor that is activated upon binding to brain-derived neurotrophic factor (BDNF). To date, the search for low-molecular-weight molecules mimicking BDNF's action has been unsuccessful. Several molecules exerting antidepressive effects in vivo, such as 7,8-DHF, have been suggested to be TrkB agonists. However, more recent publications question this hypothesis. In this study, we developed a set of experimental procedures including the evaluation of direct interactions, dimerization, downstream signaling, and cytoprotection in parallel with physicochemical and ADME methods to verify the pharmacology of 7,8-DHF and other potential reference compounds, and perform screening for novel TrkB agonists. 7,8 DHF bound to TrkB with Kd = 1.3 µM; however, we were not able to observe any other activity against the TrkB receptor in SN56 T48 and differentiated SH-SY5Y cell lines. Moreover, the pharmacokinetic and pharmacodynamic effects of 7,8-DHF at doses of 1 and 50 mg/kg were examined in mice after i.v and oral administration, respectively. The poor pharmacokinetic properties and lack of observed activation of TrkB-dependent signaling in the brain confirmed that 7,8-DHF is not a relevant tool for studying TrkB activation in vivo. The binding profile for 133 molecular targets revealed a significant lack of selectivity of 7,8-DHF, suggesting a distinct functional profile independent of interaction with TrkB. Additionally, a compound library was screened in search of novel low-molecular-weight orthosteric TrkB agonists; however, we were not able to identify reliable drug candidates. Our results suggest that published reference compounds including 7,8-DHF do not activate TrkB, consistent with canonical dogma, which indicates that the reported pharmacological activity of these compounds should be interpreted carefully in a broad functional context.

4.
Artigo em Inglês | MEDLINE | ID: mdl-34420041

RESUMO

The current guidelines for prevention of infections in hematopoietic stem cell transplantation (HSCT) do not specify which central venous catheter (CVC) insertion site should be preferred in allogeneic HSCT recipients-internal jugular vein (IJV) or subclavian vein (SCV). We designed a multicenter prospective observational study comparing the risk of infectious and non-infectious complications between the two most common sites of CVC insertion (IJV and SCV) in allogeneic HSCT. There were in total 232 consecutive patients (86 IJV and 146 SCV) who underwent adult allogeneic HSCT reported from 11 centers in 8 countries. The center independent analysis of central line associated/related blood stream infections with ECDC criteria has shown statistically significant difference favoring SCV (23% IJV vs 13% SCV (OR 2.03 (1.01-4.06), p = 0.047)). The differences in CLABSI per 1000 days of CVC use favored SCV over IJV (7.93/1000 days IJV vs 2.79/1000 days SCV, p = 0.002). The frequency of all non-infectious complications was similar in both arms-13% IJV and 12% SCV (OR 1.1 (0.5-2.5), p = 0.8). This multicenter prospective study showed statistically significant lower confirmed number of CLABSI per 1000 days of CVC use without higher risk of noninfectious complications related to the subclavian insertion site in allogeneic HSCT recipients.

5.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360938

RESUMO

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Germinação , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Plântula/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Temperatura Alta
6.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200446

RESUMO

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Temperatura Baixa , Proteoma/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteoma/análise , Proteínas Ribossômicas/genética , Ribossomos/genética
7.
EMBO J ; 40(15): e106800, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34156108

RESUMO

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

8.
Nat Commun ; 12(1): 3426, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103516

RESUMO

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/genética , Epistasia Genética , Genes de Plantas , Loci Gênicos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Hematology ; 26(1): 398-407, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34057050

RESUMO

Invasive pulmonary aspergillosis is a life-threatening complication in the cases of patients with hematologic malignancies. In December 2019, the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and the Mycoses Study Group Education and Research Consortium published a revision and an update of the consensus definitions of invasive fungal disease. The aim of this study was to evaluate the signs and radiologic patterns of early-stage invasive pulmonary aspergillosis in computed tomography in patients with hematologic entities according to the latest criteria.This retrospective analysis of a baseline high-resolution computed tomography included neutropenic patients with hematological malignancies and probable invasive pulmonary aspergillosis. The data were collected between the years 2017 and 2019. Computed tomography was performed within 72 h from the beginning of clinical symptoms: fever, dyspnea or nonproductive cough. CT scans were analyzed by two independent radiologists according to the standardized protocol based on predefined criteria.All 35 evaluated patients had typical lesions for early-stage invasive aspergillosis. Wedge-shaped infiltrates were noted in 48.6% of patients. In this group, 40% of patients had coexisting atypical radiological findings. In 11.4% of patients, wedge-shape consolidations were noted as the only type of lesions.Employment of the latest EORTC/MSG criteria increased diagnostic value of the baseline high resolution computed tomography in our study group by 11.4%.


Assuntos
Aspergillus/isolamento & purificação , Neoplasias Hematológicas/complicações , Aspergilose Pulmonar Invasiva/complicações , Aspergilose Pulmonar Invasiva/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Adulto , Idoso , Idoso de 80 Anos ou mais , Gerenciamento Clínico , Europa (Continente) , Feminino , Humanos , Aspergilose Pulmonar Invasiva/terapia , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tomografia Computadorizada por Raios X/métodos , Adulto Jovem
10.
Commun Biol ; 4(1): 181, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568709

RESUMO

Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein-metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.


Assuntos
Metabolismo Energético , Fosfoglicerato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Glicólise , Metaboloma , Metabolômica , Fosfoglicerato Quinase/genética , Mapas de Interação de Proteínas , Proteólise , Proteoma , Proteômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
Sci Rep ; 11(1): 2410, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510206

RESUMO

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10 °C, reil mutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while controlling the balance of non-translating 40S- and 60S-subunits. Reil mutations respond by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis may buffer fluctuating translation by pre-existing non-translating ribosomes before de novo synthesis meets temperature-induced demands. Reil1 reil2 double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and alter paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement of de novo synthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control of NUC2 and eIF3C2 transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.


Assuntos
Aclimatação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Ribossomos/metabolismo , Citosol/metabolismo , Mutação , Especificidade de Órgãos , Desenvolvimento Vegetal/genética , Biossíntese de Proteínas , Proteômica/métodos , Subunidades Ribossômicas/metabolismo , Transcrição Genética
12.
Autophagy ; : 1-16, 2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32967551

RESUMO

In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.

13.
Plant J ; 104(1): 7-17, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32654320

RESUMO

Plants can mitigate environmental stress conditions through acclimation. In the case of fluctuating stress conditions such as high temperatures, maintaining a stress memory enables a more efficient response upon recurring stress. In a genetic screen for Arabidopsis thaliana mutants impaired in the memory of heat stress (HS) we have isolated the FORGETTER2 (FGT2) gene, which encodes a type 2C protein phosphatase (PP2C) of the D-clade. Fgt2 mutants acquire thermotolerance normally; however, they are defective in the memory of HS. FGT2 interacts with phospholipase D α2 (PLDα2), which is involved in the metabolism of membrane phospholipids and is also required for HS memory. In summary, we have uncovered a previously unknown component of HS memory and identified the FGT2 protein phosphatase and PLDα2 as crucial players, suggesting that phosphatidic acid-dependent signaling or membrane composition dynamics underlie HS memory.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Resposta ao Choque Térmico/fisiologia , Fosfolipase D/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Resposta ao Choque Térmico/genética , Fosfolipase D/fisiologia , Fosfoproteínas Fosfatases/genética
14.
Plants (Basel) ; 9(7)2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674508

RESUMO

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

15.
Biomol NMR Assign ; 14(2): 289-293, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32583165

RESUMO

The breast cancer susceptibility protein 1 (BRCA1) plays a central role in the suppression of human breast and ovarian cancer. Germ line mutations of the BRCA1 gene are responsible for the hereditary breast and ovarian cancer (HBOC) syndrome. Here were report 1H, 13C, and 15N resonance assignments for the intrinsically disordered BRCA1 fragment 219-504, which contains important interaction sites for the proto-oncogenic transcription factor MYC as well as for p53. A nuclear magnetic resonance assignment was achieved at 18.8 T magnetic field strength using a 5D HN(CA)CONH experiment and its associated 4D H(NCA)CONH and 4D (H)N(CA)CONH experiments. 13Cα and 13Cß assignments were obtained using a 5D HabCabCONH experiment. With this strategy, 90% of 1H/15N backbone pairs could be assigned. Similarly, 264 C' resonances were assigned corresponding to 86% of the total number of C' atoms. In addition, 252 Cß resonances (i.e. 85%) were assigned, together with 461 attached Hß nuclei, as well as 264 (i.e. 86%) Cα resonances, together with 275 attached Hα nuclei.

16.
Molecules ; 25(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033464

RESUMO

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.


Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Escherichia coli/efeitos dos fármacos , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tubercidina/análogos & derivados , Tubercidina/farmacologia , 2-Aminopurina/síntese química , Acetaldeído/análogos & derivados , Acetaldeído/química , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/enzimologia , Pirimidinas/química , Tubercidina/síntese química
17.
Sci Rep ; 9(1): 11523, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395906

RESUMO

The majority of cellular processes are carried out by protein complexes. Various size fractionation methods have previously been combined with mass spectrometry to identify protein complexes. However, most of these approaches lack the quantitative information which is required to understand how changes of protein complex abundance and composition affect metabolic fluxes. In this paper we present a proof of concept approach to quantitatively study the complexome in the model plant Arabidopsis thaliana at the end of the day (ED) and the end of the night (EN). We show that size-fractionation of native protein complexes by Clear-Native-PAGE (CN-PAGE), coupled with mass spectrometry can be used to establish abundance profiles along the molecular weight gradient. Furthermore, by deconvoluting complex protein abundance profiles, we were able to drastically improve the clustering of protein profiles. To identify putative interaction partners, and ultimately protein complexes, our approach calculates the Euclidian distance between protein profile pairs. Acceptable threshold values are based on a cut-off that is optimized by a receiver-operator characteristic (ROC) curve analysis. Our approach shows low technical variation and can easily be adapted to study in the complexome in any biological system.


Assuntos
Mitocôndrias/genética , Complexos Multiproteicos/isolamento & purificação , Eletroforese em Gel de Poliacrilamida Nativa/métodos , Proteômica , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas/genética , Espectrometria de Massas , Complexos Multiproteicos/genética
18.
J Bacteriol ; 201(17)2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31235512

RESUMO

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the l-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the l-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production.IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes.


Assuntos
Coenzimas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Metaloproteínas/biossíntese , Proteínas de Escherichia coli/genética , Proteômica , Pteridinas
19.
Molecules ; 24(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995785

RESUMO

Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9--d- and N7--d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6--d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.


Assuntos
Guanina/química , Guanosina/química , Nucleosídeos/química , Purina-Núcleosídeo Fosforilase/química , Adenosina , Cinética , Nucleosídeos/análogos & derivados , Análise Espectral , Especificidade por Substrato
20.
Clin Lymphoma Myeloma Leuk ; 19(5): 264-274.e4, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30898482

RESUMO

BACKGROUND: Myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) patients, including those treated with azacitidine, are at increased risk for serious infections. The aim of our study was to identify patients with higher infectious risk at the beginning of azacitidine treatment. PATIENTS AND METHODS: We performed a retrospective evaluation of 298 MDS/CMML/AML patients and included in the analysis 232 patients who completed the first 3 cycles of azacitidine therapy or developed Grade III/IV infection before completing the third cycle. RESULTS: Overall, 143 patients (62%) experienced serious infection, and in 94 patients (41%) infection occurred within the first 3 cycles. The following variables were found to have the most significant effect on the infectious risk in multivariate analysis: red blood cell transfusion dependency (odds ratio [OR], 2.38; 97.5% confidence interval [CI], 1.21-4.79), neutropenia <0.8 × 109/L (OR, 3.03; 97.5% CI, 1.66-5.55), platelet count <50 × 109/L (OR, 2.63; 97.5% CI, 1.42-4.76), albumin level <35 g/dL (OR, 2.04; 97.5% CI, 1.01-4.16), and Eastern Cooperative Oncology Group performance status ≥2 (OR, 2.19; 97.5% CI, 1.40-3.54). Each of these variables is assigned 1 point, and the combined score represents the proposed Azacitidine Infection Risk Model. The infection rate in the first 3 cycles of therapy in lower-risk (0-2 score) and higher-risk (3-5 score) patients was 25% and 73%, respectively. The overall survival was significantly reduced in higher-risk patients compared with the lower-risk cohort (8 vs. 29 months). CONCLUSION: We selected a subset with high early risk for serious infection and worse clinical outcome among patients treated with azacitidine.


Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Azacitidina/efeitos adversos , Infecções Bacterianas/epidemiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Micoses/epidemiologia , Síndromes Mielodisplásicas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Antibioticoprofilaxia/métodos , Antifúngicos/uso terapêutico , Infecções Bacterianas/induzido quimicamente , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Feminino , Indicadores Básicos de Saúde , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Leucemia Mielomonocítica Crônica/imunologia , Leucemia Mielomonocítica Crônica/mortalidade , Masculino , Pessoa de Meia-Idade , Micoses/induzido quimicamente , Micoses/imunologia , Micoses/prevenção & controle , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/mortalidade , Polônia/epidemiologia , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Medição de Risco/métodos , Resultado do Tratamento
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