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1.
Food Chem ; 349: 129137, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33556727

RESUMO

Contamination of perfluoroalkyl substances (PFASs) in agricultural products have attracted more and more attentions recently. In this review, relationship between PFASs and vegetables is summarized comprehensively. PFASs could transfer to cultivation soils by irrigation water, bio-amended soil, and atmospheric deposition mainly from industrial emissions. Carbon chain length of PFASs, species of vegetables and so on are key factors for PFASs migration and bioaccumulation in soils, plants and vegetables. Studies on food risk assessment of PFOA and PFOS show low consumption risk for most vegetables, however researches on other substances are lacking. In the future, we need to pay more attention on novel pollution pathway in cultivation, traceability research for considerable contamination, dietary exposure levels for different vegetables and more substances, as well as more exact and scientific food risk assessments. Additionally, effective means for PFASs adsorption in soil and removal from soil are also expected.


Assuntos
Bioacumulação , Fluorcarbonetos/metabolismo , Contaminação de Alimentos/análise , Verduras/metabolismo , Alquilação , Fluorcarbonetos/química , Medição de Risco
2.
Nat Commun ; 12(1): 299, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436637

RESUMO

Chiral acetylenic derivatives are found in many bioactive compounds and are versatile functional groups in organic chemistry. Here, we describe an enantioselective nickel/Lewis acid-catalyzed asymmetric propargylic substitution reaction from simple achiral materials under mild condition. The introduction of a Lewis acid cocatalyst is crucial to the efficiency of the transformation. Notably, we investigate this asymmetric propargylic substitution reaction for the development of a range of structurally diverse natural products. The power of this strategy is highlighted by the collective synthesis of seven biologically active compounds: (-)-Thiohexital, (+)-Thiopental, (+)-Pentobarbital, (-)-AMG 837, (+)-Phenoxanol, (+)-Citralis, and (-)-Citralis Nitrile.


Assuntos
Alquinos/síntese química , Ácidos de Lewis/química , Níquel/química , Preparações Farmacêuticas/síntese química , Alquilação , Catálise , Ésteres/química , Malonatos/química , Estereoisomerismo
3.
Carbohydr Polym ; 254: 117280, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33357856

RESUMO

Polysaccharides have been used widely in many industries, from food technology and mining to cosmetics and biomedical applications. Over recent years there has been growing interest in the development of responsive polysaccharides with unique and switchable properties, particularly systems that display lower-critical solution temperatures (LCSTs). Therefore, in this study we aimed to investigate a novel strategy that would allow the conversion of non-responsive polysaccharides into thermoresponsive polysaccharides with tuneable LCSTs. Through the functionalisation of dextran with alkylamide groups (isopropyl amide, diethyl amide, piperidinyl and diisobutyl amide) using a carbodiimide coupling approach in conjunction with amic acid derivatives, we prepared a library of novel dextrans with various degrees of substitution (DS), which were characterised via nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). The alkylamide-functionalised dextrans were found to have good solubility in aqueous solutions, with the exception of those having a high DS of large hydrophobic substituents. Determination of the thermoresponsive characteristics of the polymer solutions via UV-vis spectroscopy revealed that the LCST of the alkylamide-functionalised dextrans was highly dependent on the type of alkylamide group and the DS and could be tuned over a large range (5-35 °C). Above the LCST, all of the thermoresponsive alkylamide-functionalised dextrans formed colloidal dispersions with particles sizes ranging from 400 -600 nm, as determined by dynamic light scattering (DLS). In addition, the polymers were found to exhibit a fast and reversible phase transition in solution with narrow hysteresis (∼ 1-5 °C). Finally, the injectability and biocompatibility of the novel thermoresponsive dextrans was confirmed in vivo via subcutaneous and intracranial ventricle injections, with no local or systemic toxicity noted over a 14 d period. Overall, the alkylamide-functionalised dextrans display interesting thermoresponsive properties and trends that may make them useful in biomedical applications, such as drug-delivery.


Assuntos
Polissacarídeos/química , Alquilação , Amidas/química , Animais , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Materiais Biocompatíveis/toxicidade , Cromatografia em Gel , Dextranos/administração & dosagem , Dextranos/química , Dextranos/toxicidade , Difusão Dinâmica da Luz , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peso Molecular , Ressonância Magnética Nuclear Biomolecular , Tamanho da Partícula , Transição de Fase , Polissacarídeos/administração & dosagem , Polissacarídeos/toxicidade , Espectrofotometria , Temperatura
4.
Molecules ; 26(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374613

RESUMO

A series of novel soluble nature-inspired flavin derivatives substituted with short butyl and bulky ethyl-adamantyl alkyl groups was prepared via simple and straightforward synthetic approach with moderate to good yields. The comprehensive characterization of the materials, to assess their application potential, has demonstrated that the modification of the conjugated flavin core enables delicate tuning of the absorption and emission properties, optical bandgap, frontier molecular orbital energies, melting points, and thermal stability. Moreover, the thin films prepared thereof exhibit smooth and homogeneous morphology with generally high stability over time.


Assuntos
Alquilantes/química , Riboflavina/química , Semicondutores , Alquilação
5.
PLoS One ; 15(12): e0243747, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33315907

RESUMO

Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.


Assuntos
Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , Alquilação , Indicadores e Reagentes , Luciferases de Vaga-Lume/química , Especificidade por Substrato , Temperatura
6.
Yakugaku Zasshi ; 140(11): 1313-1322, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33132266

RESUMO

In total and formal syntheses of dictyodendrins A, B, C, D, E and F, the key step involved the direct construction of the pyrrolo[2,3-c]carbazole core by the gold-catalyzed annulation of a conjugated diyne with a pyrrole to form three bonds and two aromatic rings. The subsequent introduction of substituents at the C1 (Suzuki-Miyaura coupling), C2 (acylation), N3 (alkylation) and C5 positions (Ullmann coupling) provided divergent access to dictyodendrins. Some dictyodendrin analogues exhibited inhibitory activities toward CDK2/CycA2 and GSK3.


Assuntos
Carbazóis/síntese química , Pirróis/síntese química , Acilação , Alquilação , Catálise , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Di-Inos/química , Fenômenos de Química Orgânica , Pirróis/química
7.
Nat Commun ; 11(1): 5468, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122656

RESUMO

Disulfide bonds link pairs of cysteine amino acids and their formation is assumed to be complete in the mature, functional protein. Here, we test this assumption by quantifying the redox state of disulfide bonds in the blood clotting protein fibrinogen. The disulfide status of fibrinogen from healthy human donor plasma and cultured human hepatocytes are measured using differential cysteine alkylation and mass spectrometry. This analysis identifies 13 disulfide bonds that are 10-50% reduced, indicating that fibrinogen is produced in multiple disulfide-bonded or covalent states. We further show that disulfides form upon fibrin polymerization and are required for a robust fibrin matrix that withstands the mechanical forces of flowing blood and resists premature fibrinolysis. The covalent states of fibrinogen are changed by fluid shear forces ex vivo and in vivo, indicating that the different states are dynamic. These findings demonstrate that fibrinogen exists and functions as multiple covalent forms.


Assuntos
Dissulfetos/química , Fibrinogênio/química , Trombose/metabolismo , Alquilação , Testes de Coagulação Sanguínea , Fibrina/biossíntese , Fibrinólise , Hepatócitos , Humanos , Espectrometria de Massas , Oxirredução , Polimerização , Trombina/metabolismo
8.
Nat Commun ; 11(1): 4722, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948764

RESUMO

Precisely controlled deuterium labeling at specific sites of N-alkyl drugs is crucial in drug-development as over 50% of the top-selling drugs contain N-alkyl groups, in which it is very challenging to selectively replace protons with deuterium atoms. With the goal of achieving controllable isotope-labeling in N-alkylated amines, we herein rationally design photocatalytic water-splitting to furnish [H] or [D] and isotope alkanol-oxidation by photoexcited electron-hole pairs on a polymeric semiconductor. The controlled installation of N-CH3, -CDH2, -CD2H, -CD3, and -13CH3 groups into pharmaceutical amines thus has been demonstrated by tuning isotopic water and methanol. More than 50 examples with a wide range of functionalities are presented, demonstrating the universal applicability and mildness of this strategy. Gram-scale production has been realized, paving the way for the practical photosynthesis of pharmaceuticals.


Assuntos
Aminas/química , Aminas/metabolismo , Luz , Semicondutores , Alquilação , Aminas/farmacologia , Catálise , Deutério , Oxirredução , Preparações Farmacêuticas , Prótons , Água , Difração de Raios X
9.
PLoS One ; 15(8): e0228525, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822344

RESUMO

The toxic effect of strained hydrocarbon 2,2'-bis (bicyclo[2.2.1]heptane) (BBH) was studied using whole-cell bacterial lux-biosensors based on Escherichia coli cells in which luciferase genes are transcriptionally fused with stress-inducible promoters. It was shown that BBH has the genotoxic effect causing bacterial SOS response however no alkylating effect has been revealed. In addition to DNA damage, there is an oxidative effect causing the response of OxyR/S and SoxR/S regulons. The most sensitive to BBH lux-biosensor was E. coli pSoxS-lux which reacts to the appearance of superoxide anion radicals in the cell. It is assumed that the oxidation of BBH leads to the generation of reactive oxygen species, which provide the main contribution to the genotoxicity of this substance.


Assuntos
Compostos Bicíclicos com Pontes/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Mutagênicos/toxicidade , Alquilação/efeitos dos fármacos , Técnicas Biossensoriais , Dano ao DNA , Relação Dose-Resposta a Droga , Escherichia coli/citologia , Escherichia coli/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Regulon/efeitos dos fármacos , Regulon/genética
10.
J Vis Exp ; (160)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32628175

RESUMO

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.


Assuntos
Espectrometria de Massas/métodos , Ubiquitina/análise , Alquilação , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Leupeptinas/farmacologia , Lisina/metabolismo , Camundongos , Peptídeos/análise , Proteoma , Padrões de Referência , Software , Ubiquitina/química
11.
Sci Rep ; 10(1): 10958, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616737

RESUMO

Road runoff carries a mixture of contaminants that threatens the quality of natural water bodies and the health of aquatic organisms. The use of sedimentation ponds is a nature-based solution for the treatment of road runoff. This study assessed the concentration of polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in sediment from seven highway sedimentation ponds and three natural urban ponds. In addition, the study explored the bioaccumulation of PAHs in dragonfly nymphs (Anisoptera). Finally, biota-sediment accumulation factors (BSAFs) were estimated. The results revealed a significant difference in the concentrations of 16 priority PAHs in sediment, with overall higher levels in sedimentation ponds (2,911 µg/kg on average) compared to natural urban ponds (606 µg/kg on average). PAH levels increased substantially once alkylated homologues were considered, with alkylated comprising between 42 and 87% of the total PAH in sediment samples. These results demonstrate the importance of alkylated forms in the environmental assessment of PAHs. The bioaccumulation assessment indicates that dragonfly nymphs bioaccumulate PAHs to a certain degree. It is not clear, however, whether they metabolize PAHs. BSAF results ranged from approx. 0.006 to 10 and indicate that BSAFs can be a powerful tool to determine the functionality of sedimentation ponds.


Assuntos
Alquilantes/química , Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Ninfa/metabolismo , Odonatos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/análise , Poluentes Químicos da Água/análise , Alquilação , Animais , Bioacumulação , Sedimentos Geológicos/química , Ninfa/crescimento & desenvolvimento , Odonatos/crescimento & desenvolvimento , Hidrocarbonetos Policíclicos Aromáticos/química , Poluentes Químicos da Água/química
12.
Nature ; 584(7819): 69-74, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32512577

RESUMO

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.


Assuntos
Alcenos/química , Alcenos/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Luz , Oxirredutases/metabolismo , Processos Fotoquímicos/efeitos da radiação , Álcoois/química , Álcoois/metabolismo , Alquilação/efeitos da radiação , Biocatálise/efeitos da radiação , Biomassa , Carboxiliases/metabolismo , Flavinas/metabolismo , Modelos Químicos , Modelos Moleculares , Estereoisomerismo
13.
Nat Commun ; 11(1): 2756, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488003

RESUMO

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.


Assuntos
Etanol/análogos & derivados , Etanol/química , Compostos de Organossilício/química , Trifluoretanol/química , Álcoois/química , Alquilação , Técnicas de Química Sintética , Indicadores e Reagentes/química , Estrutura Molecular
14.
Nat Chem Biol ; 16(8): 904-911, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32483377

RESUMO

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.


Assuntos
Antibacterianos/química , Fosfato de Piridoxal/química , Alquilação/fisiologia , Antibacterianos/farmacologia , Fenômenos Bioquímicos , Metionina/metabolismo , Nucleosídeos/química , Fosfatos , Fosforilação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química
15.
Proc Natl Acad Sci U S A ; 117(17): 9318-9328, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32273391

RESUMO

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.


Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças Ópticas
16.
Nature ; 581(7809): 415-420, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32268340

RESUMO

The ubiquity of tertiary alkylamines in pharmaceutical and agrochemical agents, natural products and small-molecule biological probes1,2 has stimulated efforts towards their streamlined synthesis3-9. Arguably the most robust method for the synthesis of tertiary alkylamines is carbonyl reductive amination3, which comprises two elementary steps: the condensation of a secondary alkylamine with an aliphatic aldehyde to form an all-alkyl-iminium ion, which is subsequently reduced by a hydride reagent. Direct strategies have been sought for a 'higher order' variant of this reaction via the coupling of an alkyl fragment with an alkyl-iminium ion that is generated in situ10-14. However, despite extensive efforts, the successful realization of a 'carbonyl alkylative amination' has not yet been achieved. Here we present a practical and general synthesis of tertiary alkylamines through the addition of alkyl radicals to all-alkyl-iminium ions. The process is facilitated by visible light and a silane reducing agent, which trigger a distinct radical initiation step to establish a chain process. This operationally straightforward, metal-free and modular transformation forms tertiary amines, without structural constraint, via the coupling of aldehydes and secondary amines with alkyl halides. The structural and functional diversity of these readily available precursors provides a versatile and flexible strategy for the streamlined synthesis of complex tertiary amines.


Assuntos
Aminas/química , Aminas/síntese química , Técnicas de Química Sintética/métodos , Aldeídos/química , Alquilação , Aminação , Loratadina/análogos & derivados , Loratadina/síntese química , Loratadina/química
17.
Chem Commun (Camb) ; 56(26): 3741-3744, 2020 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-32124910

RESUMO

Continuous efforts have been invested in the selective modification of proteins. Herein, we first report the construction of sulfonium tethered cyclic peptides via an intramolecular cyclization by an aliphatic halide. This cyclization could enhance the stability and cellular uptake of peptides. Furthermore, the sulfonium center could be recognized by cysteine in the vicinity of the protein-peptide interacting interface and form a peptide-protein conjugate.


Assuntos
Metionina/química , Peptídeos Cíclicos/química , Compostos de Sulfônio/química , Alquilação , Transporte Biológico , Ciclização , Células HeLa , Humanos , Peptídeos Cíclicos/farmacologia
18.
Biomed Khim ; 66(1): 18-29, 2020 Jan.
Artigo em Russo | MEDLINE | ID: mdl-32116223

RESUMO

Mass spectrometric proteomic analysis at the sample preparation stage involves the artificial reduction of disulfide bonds in proteins formed between cysteine residues. Such bonds, when preserved in their native state, complicate subsequent enzymatic hydrolysis and interpretation of the research results. To prevent the re-formation of the disulfide bonds, cysteine residues are protected by special groups, most often by alkylation. In this review, we consider the methods used to modify cysteine residues during sample preparation, as well as possible artifacts of this stage. Particularly, adverse reactions of the alkylating agents with other amino acid residues are described. The most common alkylating compound used to protect cysteine residues in mass spectrometric proteomic analysis is iodoacetamide. However, an analysis of the literature in this area indicates that this reagent causes more adverse reactions than other agents used, such as chloroacetamide and acrylamide. The latter can be recommended for wider use. In the review we also discuss the features of the cysteine residue modifications and their influence on the efficiency of the search for post-translational modifications and protein products of single nucleotide substitutions.


Assuntos
Artefatos , Cisteína/química , Espectrometria de Massas , Proteômica , Alquilação
19.
Nat Commun ; 11(1): 1458, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193394

RESUMO

Esterification reactions are central to many aspects of industrial and biological chemistry. The formation of carboxyesters typically occurs through nucleophilic attack of an alcohol onto the carboxylate carbon. Under certain conditions employed in organic synthesis, the carboxylate nucleophile can be alkylated to generate esters from alkyl halides, but this reaction has only been observed transiently in enzymatic chemistry. Here, we report a carboxylate alkylating enzyme - BrtB - that catalyzes O-C bond formation between free fatty acids of varying chain length and the secondary alkyl halide moieties found in the bartolosides. Guided by this reactivity, we uncovered a variety of natural fatty acid-bartoloside esters, previously unrecognized products of the bartoloside biosynthetic gene cluster.


Assuntos
Proteínas de Bactérias/metabolismo , Synechocystis/enzimologia , Transferases/metabolismo , Alquilantes , Alquilação , Proteínas de Bactérias/genética , Técnicas de Química Sintética/métodos , Esterificação , Ésteres/metabolismo , Ácidos Graxos/metabolismo , Família Multigênica , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Synechocystis/genética , Transferases/genética
20.
BMC Biol ; 18(1): 32, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209106

RESUMO

BACKGROUND: Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy. RESULTS: Using streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-ß. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis. CONCLUSIONS: These results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.


Assuntos
RNA Helicases DEAD-box/genética , NF-kappa B/metabolismo , Receptores de Reconhecimento de Padrão/genética , Transdução de Sinais , Alquilação , Animais , RNA Helicases DEAD-box/metabolismo , Sondas de DNA , Tratamento Farmacológico , Humanos , Camundongos , Receptores de Reconhecimento de Padrão/metabolismo
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