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Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.
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Formaldehyde is a pollutant and human metabolite that is toxic at high concentrations. Biological studies on formaldehyde are hindered by its high reactivity and volatility, which make it challenging to deliver quantitatively to cells. Here, we describe the development and validation of a set of N-acyloxymethyl-phthalimides as cell-relevant formaldehyde delivery agents. These esterase-sensitive compounds were similarly or less inhibitory to human cancer cell growth than free formaldehyde but the lead compound increased intracellular formaldehyde concentrations, increased cellular levels of thymidine derivatives (implying increased formaldehyde-mediated carbon metabolism), induced formation of cellular DNA-protein cross-links and induced cell death in pancreatic cancer cells. Overall, our N-acyloxymethyl-phthalimides and control compounds provide an accessible and broadly applicable chemical toolkit for formaldehyde biological research and have potential as cancer therapeutics.
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Formaldehyde (HCHO) is a toxic and carcinogenic pollutant and human metabolite that reacts with biomolecules under physiological conditions. Quantifying HCHO is essential for ongoing biological and biomedical research on HCHO; however, its reactivity, small size and volatility make this challenging. Here, we report a novel HCHO detection/quantification method that couples cysteamine-mediated HCHO scavenging with SPME GC-MS analysis. Our NMR studies confirm cysteamine as an efficient and selective HCHO scavenger that out-competes O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, the most commonly used scavenger, and forms a stable thiazolidine amenable to GC-MS quantification. Validation of our GC-MS method using FDA and EMA guidelines revealed detection and quantification limits in the nanomolar and micromolar ranges respectively, while analysis of bacterial cell lysate confirmed its applicability in biological samples. Overall, our studies confirm that cysteamine scavenging coupled to SPME GC-MS analysis provides a sensitive and chemically robust method to quantify HCHO in biological samples.
Assuntos
Pesquisa Biomédica , Cisteamina , Humanos , Cromatografia Gasosa-Espectrometria de Massas , Microextração em Fase Sólida , FormaldeídoRESUMO
Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.
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Transient receptor potential (TRP) channels have important roles in environmental sensing in animals. Human TRP subfamily A member 1 (TRPA1) is responsible for sensing allyl isothiocyanate (AITC) and other electrophilic sensory irritants. TRP subfamily vanilloid member 3 (TRPV3) is involved in skin maintenance. TRPV3 is a reported substrate of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH). We report biochemical and structural studies concerning asparaginyl hydroxylation of the ankyrin repeat domains (ARDs) of TRPA1 and TRPV3 catalysed by FIH. The results with ARD peptides support a previous report on FIH-catalysed TRPV3 hydroxylation and show that, of the 12 potential TRPA1 sequences investigated, one sequence (TRPA1 residues 322-348) undergoes hydroxylation at Asn336. Structural studies reveal that the TRPA1 and TRPV3 ARDs bind to FIH with a similar overall geometry to most other reported FIH substrates. However, the binding mode of TRPV3 to FIH is distinct from that of other substrates.
Assuntos
Repetição de Anquirina , Síndrome do Desconforto Respiratório , Humanos , Animais , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Hidroxilação , Oxigenases de Função Mista/metabolismo , Ligação Proteica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Tetrahydrofolic acid and formaldehyde are key human metabolites but their physiologically relevant chemistry is undefined. Our NMR studies confirm formaldehyde as a product of tetrahydrofolic acid degradation but also reveal their reaction regulates the stability of tetrahydrofolic acid. These observations identify a novel non-enzymatic feedback mechanism regulating formaldehyde and folate metabolism that has important implications for folate-targeting chemotherapy in cancer and other diseases.
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Formaldeído/metabolismo , Tetra-Hidrofolatos/metabolismo , Timidilato Sintase/metabolismo , Biocatálise , Estabilidade Enzimática , Formaldeído/química , Humanos , Estrutura Molecular , Tetra-Hidrofolatos/química , Timidilato Sintase/químicaRESUMO
Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.
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Hipóxia Celular , Código das Histonas , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/antagonistas & inibidores , Quelantes de Ferro/toxicidade , Células MCF-7 , Processamento de Proteína Pós-TraducionalRESUMO
Metampicillin is a ß-lactam antibiotic that is prepared by the reaction of ampicillin with formaldehyde. Although metampicillin has been studied for treatment of infections in animals and humans, its structure has been unclear. We report NMR studies revealing that metampicillin contains a formaldehyde-derived cyclic aminal. NMR time-course experiments with excess formaldehyde in solution show formation of another product with an additional exocyclic hemiaminal group formed by reaction with the cyclic aminal nitrogen. The exocyclic hemiaminal group is readily removed by reaction with the formaldehyde scavenger 1,3-cyclohexanedione, whereas the cyclic aminal methylene exhibits greater stability. The overall results assign the structure of metampicillin as containing a cyclic aminal and further reveal the potential for complexity in the reaction of formaldehyde with biomedicinally relevant molecules.
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Formaldehyde (HCHO) is a simple and highly reactive human metabolite but its biochemistry is poorly defined. A limiting factor in HCHO research is lack of validated quantification methods for HCHO relevant to biological samples. We describe spectroscopic studies on a reported fluorescence-based HCHO detection method involving its reaction with ampicillin. The results validate the structure and fluorescence properties of the HCHO-ampicillin reaction product. However, the same adduct is observed after reaction of ampicillin with glyoxylate. Related fluorophores were formed with other biologically relevant carbonyl compounds. Overall, our studies suggest the ampicillin method is not reliable for selective detection and quantification of HCHO in biological samples.
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AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
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Proteínas de Ligação ao Cálcio/química , Proteínas de Membrana/química , Oxigenases de Função Mista/química , Proteínas Musculares/química , Sequência de Aminoácidos , Asparagina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Cristalografia , Dissulfetos/química , Dissulfetos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Conformação ProteicaRESUMO
Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-K mO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein-coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.
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Dioxigenases/metabolismo , Oxigênio/metabolismo , Anaerobiose , Arabidopsis/genética , Arabidopsis/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Cisteína/metabolismo , Dioxigenases/genética , Humanos , Interleucinas/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Proteínas RGS/metabolismoRESUMO
Horizontal gene transfer (HGT) mediated by the spread of plasmids fuels evolution in prokaryotes. Although plasmids provide bacteria with new adaptive genes, they also produce physiological alterations that often translate into a reduction in bacterial fitness. The fitness costs associated with plasmids represent an important limit to plasmid maintenance in bacterial communities, but their molecular origins remain largely unknown. In this work, we combine phenomics, transcriptomics and metabolomics to study the fitness effects produced by a collection of diverse plasmids in the opportunistic pathogen Pseudomonas aeruginosa PAO1. Using this approach, we scan the physiological changes imposed by plasmids and test the generality of some main mechanisms that have been proposed to explain the cost of HGT, including increased biosynthetic burden, reduced translational efficiency, and impaired chromosomal replication. Our results suggest that the fitness effects of plasmids have a complex origin, since none of these mechanisms could individually provide a general explanation for the cost of plasmid carriage. Interestingly, our results also showed that plasmids alter the expression of a common set of metabolic genes in PAO1, and produce convergent changes in host cell metabolism. These surprising results suggest that there is a common metabolic response to plasmids in P. aeruginosa PAO1.
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Replicação do DNA , DNA Bacteriano/genética , Plasmídeos/genética , Pseudomonas aeruginosa/genética , Transferência Genética Horizontal , Aptidão Genética , Pseudomonas aeruginosa/fisiologiaRESUMO
N-Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)-catalysed demethylation of Nε -methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A-catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM-catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells.
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Desmetilação , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Histonas/química , Histonas/genética , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/genética , Cinética , Lisina/química , Lisina/genética , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Jumonji domain-containing demethylases (JmjC-KDMs) catalyse demethylation of Nε-methylated lysines on histones and play important roles in gene regulation. We report selectivity studies on KDM6B (JMJD3), a disease-relevant JmjC-KDM, using synthetic lysine analogues. The results unexpectedly reveal that KDM6B accepts multiple Nε-alkylated lysine analogues, forming alcohol, aldehyde and carboxylic acid products.
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Histona Desmetilases com o Domínio Jumonji/metabolismo , Sequência de Aminoácidos , Biocatálise , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Lisina/metabolismo , Oxirredução , Peptídeos/síntese química , Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors.
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Sistemas de Liberação de Medicamentos , Histona Desmetilases/química , Histonas/química , Bibliotecas de Moléculas Pequenas/síntese química , Amiodarona/química , Avaliação Pré-Clínica de Medicamentos , Lisina/química , Estrutura Molecular , Filogenia , Proteínas de Plantas/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The originally published version of this Article contained an error in the spelling of the author Md. Saiful Islam, which was incorrectly given as Saiful Islam. This has now been corrected in both the PDF and HTML versions of the Article.
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Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.
Assuntos
Arginina/química , Proteínas de Transporte/química , Histona Desmetilases/química , Proteínas de Membrana/química , Proteína S6 Ribossômica/química , Arginina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Hidroxilação , Cinética , Lisina/química , Lisina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Estereoisomerismo , Especificidade por Substrato , TermodinâmicaRESUMO
2-Oxoglutarate (2OG)-dependent oxygenases (2OGXs) catalyze a remarkably diverse range of oxidative reactions. In animals, these comprise hydroxylations and N-demethylations proceeding via hydroxylation; in plants and microbes, they catalyze a wider range including ring formations, rearrangements, desaturations, and halogenations. The catalytic flexibility of 2OGXs is reflected in their biological functions. After pioneering work identified the roles of 2OGXs in collagen biosynthesis, research revealed they also function in plant and animal development, transcriptional regulation, nucleic acid modification/repair, fatty acid metabolism, and secondary metabolite biosynthesis, including of medicinally important antibiotics. In plants, 2OGXs are important agrochemical targets and catalyze herbicide degradation. Human 2OGXs, particularly those regulating transcription, are current therapeutic targets for anemia and cancer. Here, we give an overview of the biochemistry of 2OGXs, providing examples linking to biological function, and outline how knowledge of their enzymology is being exploited in medicine, agrochemistry, and biocatalysis.
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Ácidos Cetoglutáricos/metabolismo , Oxigenases/metabolismo , Animais , Biocatálise , Colágeno/biossíntese , Humanos , Hidroxilação , Modelos Biológicos , Modelos Moleculares , Oxirredução , Oxigenases/química , Conformação Proteica , Especificidade por SubstratoRESUMO
Prolyl hydroxylation of hypoxia inducible factor (HIF)-α, as catalysed by the Fe(ii)/2-oxoglutarate (2OG)-dependent prolyl hydroxylase domain (PHD) enzymes, has a hypoxia sensing role in animals. We report that binding of prolyl-hydroxylated HIF-α to PHD2 is â¼50 fold hindered by prior 2OG binding; thus, when 2OG is limiting, HIF-α degradation might be inhibited by PHD binding.