Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34.294
Filtrar
1.
Nat Commun ; 12(1): 5771, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599190

RESUMO

Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1-deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.


Assuntos
Elementos Facilitadores Genéticos , Células Germinativas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Animais , Diferenciação Celular , Cromatina/metabolismo , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Masculino , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/citologia , Mutação/genética , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição , Transcrição Genética
2.
J Agric Food Chem ; 69(41): 12333-12343, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34633809

RESUMO

Memory impairment is becoming a potential health issue with the delicacy of diet and social stress. Sea cucumber peptides (SCP) prevent memory impairment, as previously reported. In this study, further research was performed using hippocampal lysine-acetylome to explore molecular regulation mechanisms. C57BL/6 mice were treated with scopolamine via intraperitoneal injection to simulate memory impairment. To determine the influence of SCP on the total acetylated-protein level of the hippocampus, acetylated-proteomics was performed. SCP increased the acetylation level of histone (H3 and H4). Meanwhile, for non-histones, the differentially acetylated proteins were involved in multiple memory-related pathways, as shown by KEGG enrichment analysis. Additionally, long-term potentiation was confirmed by western blotting. Finally, a combined analysis of proteome and lysine acetylome revealed that SCP contributed to synaptic vesicle cycle regulation and dopamine metabolism. Consequently, our findings revealed that SCP was potentially neuroprotective by regulating post-transcriptional hippocampal protein acetylation.


Assuntos
Lisina , Pepinos-do-Mar , Acetilação , Animais , Hipocampo/metabolismo , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos , Processamento de Proteína Pós-Traducional , Proteoma/genética , Proteoma/metabolismo , Pepinos-do-Mar/metabolismo
3.
Nat Commun ; 12(1): 5293, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489448

RESUMO

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , DNA Bacteriano/química , DnaB Helicases/química , Helicobacter pylori/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/química , Compostos de Alumínio/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fluoretos/química , Fluoretos/metabolismo , Expressão Gênica , Helicobacter pylori/genética , Hidrólise , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
4.
Front Cell Infect Microbiol ; 11: 679792, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34568085

RESUMO

Binding to plasminogen (Plg) enables bacteria to associate with and invade host tissues. The cell wall protein PbsP significantly contributes to the ability of group B streptococci, a frequent cause of invasive infection, to bind Plg. Here we sought to identify the molecular regions involved in the interactions between Plg and PbsP. The K4 Kringle domain of the Plg molecule was required for binding of Plg to whole PbsP and to a PbsP fragment encompassing a region rich in methionine and lysine (MK-rich domain). These interactions were inhibited by free L-lysine, indicating the involvement of lysine binding sites in the Plg molecule. However, mutation to alanine of all lysine residues in the MK-rich domain did not decrease its ability to bind Plg. Collectively, our data identify a novel bacterial sequence that can interact with lysine binding sites in the Plg molecule. Notably, such binding did not require the presence of lysine or other positively charged amino acids in the bacterial receptor. These data may be useful for developing alternative therapeutic strategies aimed at blocking interactions between group B streptococci and Plg.


Assuntos
Lisina , Plasminogênio , Sítios de Ligação , Parede Celular/metabolismo , Lisina/metabolismo , Plasminogênio/metabolismo , Ligação Proteica , Streptococcus agalactiae
5.
MAbs ; 13(1): 1974150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34486490

RESUMO

This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an IgG1 antibody and a duocarmycin analog-based payload-linker. Of the 80 putative lysine sites, 78 were found to be conjugated via tryptic peptide mapping and LC-HRMS. Surprisingly, seven cysteine-linked conjugated peptides were also detected resulting from the conjugation of cysteine residues derived from the four inter-chain disulfide bonds during the reaction. This unexpected finding could be attributed to the free thiols of the 2-IT thiolated antibody intermediates and/or the 4-mercaptobutanamide by-product resulting from the hydrolysis of 2-IT. These free thiols could cause the four inter-chain disulfide bonds of the antibody to scramble via intra- or inter-molecular attack. The presence of only pair of non-reactive (unconjugated) lysine residues, along with the four intact intra-chain disulfide bonds, is attributed to their poor accessibility, which is consistent with solvent accessibility modeling analysis. We also discovered a major by-product derived from the hydrolysis of the amidine moiety of the N-terminus conjugate. In contrast, the amidine moiety in lysine-linked conjugates appeared stable. Based on our results, we propose plausible formation mechanisms of cysteine-linked conjugates and the hydrolysis of the N-terminus conjugate, which provide scientific insights that are beneficial to process development and drug quality control.


Assuntos
Cisteína/química , Descoberta de Drogas/métodos , Imunoconjugados/química , Lisina/química , Duocarmicinas/análogos & derivados , Humanos , Imunoglobulina G/química
6.
Nat Commun ; 12(1): 5548, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545082

RESUMO

Isoniazid (INH) is a first-line anti-tuberculosis drug used for nearly 70 years. However, the mechanism underlying the side effects of INH has remained elusive. Here, we report that INH and its metabolites induce a post-translational modification (PTM) of histones, lysine isonicotinylation (Kinic), also called 4-picolinylation, in cells and mice. INH promotes the biosynthesis of isonicotinyl-CoA (Inic-CoA), a co-factor of intracellular isonicotinylation. Mass spectrometry reveals 26 Kinic sites in histones in HepG2 cells. Acetyltransferases CREB-binding protein (CBP) and P300 catalyse histone Kinic, while histone deacetylase HDAC3 functions as a deisonicotinylase. Notably, MNase sensitivity assay and RNA-seq analysis show that histone Kinic relaxes chromatin structure and promotes gene transcription. INH-mediated histone Kinic upregulates PIK3R1 gene expression and activates the PI3K/Akt/mTOR signalling pathway in liver cancer cells, linking INH to tumourigenicity in the liver. We demonstrate that Kinic is a histone acylation mark with a pyridine ring, which may have broad biological effects. Therefore, INH-induced isonicotinylation potentially accounts for the side effects in patients taking INH long-term for anti-tuberculosis therapy, and this modification may increase the risk of cancer in humans.


Assuntos
Antituberculosos/farmacologia , Código das Histonas , Isoniazida/farmacologia , Ácidos Isonicotínicos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Coenzima A/metabolismo , Células HeLa , Células Hep G2 , Histona Desacetilases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Ácidos Isonicotínicos/química , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismo
7.
Nat Commun ; 12(1): 5277, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489474

RESUMO

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.


Assuntos
Proteínas de Escherichia coli/química , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/química , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Complexo Piruvato Desidrogenase/isolamento & purificação , Ácido Tióctico/análogos & derivados , Ácido Tióctico/química , Ácido Tióctico/metabolismo
8.
Biomater Sci ; 9(20): 6865-6878, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-34494620

RESUMO

Despite the development of advanced tissue engineering substitutes, inflammation is still a significant problem that can arise from inflamed burn injuries, chronic wounds, or microbial diseases. Although topical wound dressing accelerates healing by minimizing or preventing the consequences of skin inflammation, there remains a need for the development of a novel substitute scaffold that can effectively eliminate immoderate inflammation and infection in the initial phase of the healing meachanism. In this study, an artificial skin substitute scaffold fabricated with asiaticoside (AS) and epsilon-poly-L-lysine (εPLL) was prepared. Upon the release of these bioactive compounds, they accelerate wound healing and inhibit any bacterial infection at the wound site. We determined whether AS and εPLL exhibit anti-inflammatory and bactericidal effects through different mechanisms. Collectively, the collagen-AS/εPLL artificial skin substitute could be a significant therapeutic agent for scar-less rapid wound healing (without infection and inflammation) of initially-inflamed full-thickness wounds.


Assuntos
Lisina , Cicatrização , Anti-Inflamatórios/farmacologia , Colágeno , Triterpenos
9.
Soft Matter ; 17(37): 8459-8464, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34494056

RESUMO

Exposure of lysine-containing peptide-based gelators to the cross-linking agent glutaraldehyde allows tuning of gel mechanical properties. The effect of cross-linking depends on the position of the lysine residue in the peptide chain, the concentration of gelator and the conditions under which cross-linking takes place. Through control of these factors, cross-linking leads to increased gel strength.


Assuntos
Hidrogéis , Lisina , Reagentes para Ligações Cruzadas , Glutaral , Peptídeos
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(9): 808-814, 2021 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-34533128

RESUMO

Objective To investigate the effect of lysine-specific demethylase 3A (KDM3A) on the invasion and migration of MDA-MB-231 breast cancer cells. Methods The mRNA and the protein expressions of KDM3A in MDA-MB-231 breast cancer cells and MCF-10A normal breast cells were detected by real-time quantitative PCR and Western blotting, respectively; the KDM3A level of MDA-MB-231 cells was knocked down by lentivirus infection of KDM3A short hairpin RNA (shKDM3A). The change of invasion and migration ability of MDA-MB-231 cells was detected by TranswellTM assay, and the change in the cell cycle was detected by flow cytometry. Results The expression of KDM3A in MDA-MB-231 breast cancer cells was significantly increased compared with that in MCF-10A epithelial cells; after KDM3A knockdown, the invasion and migration abilities of MDA-MB-231 cells were significantly decreased, and the cell cycle was arrested in the G0/G1 phase. Conclusion Knockdown of KDM3A inhibits the invasion and migration of MDA-MB-231 breast cancer cells and arrests the cell cycle in G0/G1 phase.


Assuntos
Neoplasias da Mama , Lisina , Neoplasias da Mama/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Fase G1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Invasividade Neoplásica/genética
11.
Nat Cell Biol ; 23(9): 978-991, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34497368

RESUMO

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Poliubiquitina/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Genes Supressores de Tumor/fisiologia , Humanos , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo
12.
J Agric Food Chem ; 69(40): 11960-11970, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34591478

RESUMO

Despite more than 100 years of research, formation of food melanoidins from carbohydrates and amino acids in the course of the Maillard reaction is still not fully understood. Experiments with relevant precursors are commonly used to limit the pathways of the complex reaction and to elucidate the formation mechanisms of the colored end-products. Here as a simple model, methylglyoxal was incubated with l-alanine or l-lysine in aqueous solutions at 100 °C and pH 5. The reaction mixtures were analyzed for color formation, molecular weight distribution, and conversion of methylglyoxal. High-resolution mass spectrometry was used to characterize the variety of products formed. With the help of Kendrick and van Krevelen analyses, the complex data sets were investigated for common substructures and reaction patterns. This study revealed that methylglyoxal forms oligomers via aldol reaction under involvement of its prevalent reaction products such as formaldehyde, acetaldehyde, acetol, and aminoacetone with amino acids.


Assuntos
Lisina , Reação de Maillard , Alanina , Espectrometria de Massas , Polímeros , Aldeído Pirúvico
13.
Microb Pathog ; 160: 105169, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34509528

RESUMO

Vibrio parahaemolyticus is one of the most common pathogenic bacteria that pose a threat to human health. The purpose of this study was to investigate antibacterial mechanisms of ε-poly-lysine (ε-PL) against V. parahaemolyticus using a lable free-based proteomic analysis. The differentially expressed proteins (DEPs) were subjected to bioinformatics analysis. The results indicated that a total of 196 DEPs, including 118 up-regulated and 78 down-regulated, were identified in the ε-PL-treated cells compared with control group. Upon Go functional enrichment, 13, 9, and 8 specific Go terms in biological processes, molecular functions and cellular components were identified, respectively. KEGG pathways analysis indicated that the DEPs were mainly involved in bacterial chemotaxis, RNA transport and two-component system, which were significantly enriched (P < 0.05). In PPI analysis, Che R and Che V, both involved in bacterial chemotaxis and RNA transport pathways, are closely related to other DEPs. Therefore, the down-regulation of Che R and Che V in ε-PL-treated cells resulted in the reduction or even loss of bacterial adaptability, and they were the critical action sites of ε-PL to inactivate V. parahaemolyticus.


Assuntos
Anti-Infecciosos , Vibrio parahaemolyticus , Antibacterianos , Humanos , Lisina , Proteômica
14.
Nat Commun ; 12(1): 4800, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417450

RESUMO

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.


Assuntos
Cromatina/metabolismo , Genes Essenciais , Histonas/metabolismo , Lisina/metabolismo , Transcrição Genética , Sequência de Aminoácidos , Animais , Ciclo Celular/genética , Linhagem Celular , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Espectroscopia de Ressonância Magnética , Metilação , Camundongos , Modelos Biológicos , Nucleossomos/metabolismo , Conformação Proteica
15.
Biomolecules ; 11(8)2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34439834

RESUMO

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.


Assuntos
Glutaratos/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Succinatos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Encéfalo/metabolismo , Cromatografia de Afinidade , Soros Imunes/química , Immunoblotting , Fígado/metabolismo , Masculino , Coelhos , Ratos
16.
Molecules ; 26(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34443518

RESUMO

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.


Assuntos
Produtos Biológicos/química , Lisina/análogos & derivados , Myxococcales/química , Niacina/química , Vias Biossintéticas/genética , Genoma Bacteriano/genética , Lisina/química , Metaboloma/genética , Myxococcales/genética , Myxococcales/isolamento & purificação , Niacina/isolamento & purificação
17.
Appl Microbiol Biotechnol ; 105(18): 6899-6908, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34455479

RESUMO

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. KEY POINTS: • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.


Assuntos
Aspartoquinase Homosserina Desidrogenase , Escherichia coli , Aminoácidos , Escherichia coli/genética , Lisina , Temperatura
18.
Biomolecules ; 11(7)2021 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-34356675

RESUMO

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Epigênese Genética , Humanos , Lisina/química , Lisina/metabolismo , Simulação de Dinâmica Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerização Proteica , Prótons , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tirosina/química , Tirosina/metabolismo
19.
Biophys J ; 120(17): 3615-3627, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34339634

RESUMO

The RNA-dependent RNA polymerase (RdRp) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a promising drug target for coronavirus disease 2019 (COVID-19) because it plays the most important role in the replication of the RNA genome. Nucleotide analogs such as remdesivir and favipiravir are thought to interfere with the RNA replication by RdRp. More specifically, they are expected to compete with nucleoside triphosphates, such as ATP. However, the process in which these drug molecules and nucleoside triphosphates are taken up by RdRp remains unknown. In this study, we performed all-atom molecular dynamics simulations to clarify the recognition mechanism of RdRp for these drug molecules and ATP that were at a distance. The ligand recognition ability of RdRp decreased in the order of remdesivir, favipiravir, and ATP. We also identified six recognition paths. Three of them were commonly found in all ligands, and the remaining three paths were ligand-dependent ones. In the common two paths, it was observed that the multiple lysine residues of RdRp carried the ligands to the binding site like a "bucket brigade." In the remaining common path, the ligands directly reached the binding site. Our findings contribute to the understanding of the efficient ligand recognition by RdRp at the atomic level.


Assuntos
COVID-19 , RNA Polimerase Dependente de RNA , Antivirais , Humanos , Lisina , SARS-CoV-2
20.
J Dairy Sci ; 104(10): 10753-10779, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34364648

RESUMO

The effects of dietary fatty acid (FA) and starch content as well as supplemental digestible Lys (sdLys) on production, energy utilization, and N utilization were evaluated. Each factor was fed at 5 different amounts, and factor limits were as follows: 3.0 to 6.2% of dry matter (DM) for FA; 20.2 to 31.3% of DM for starch, and 0 to 17.8 g/d of sdLys. Dietary FA and starch were increased by replacing soyhulls with supplemental fat and corn grain, respectively, and sdLys increased with rumen-protected Lys. Fifteen unique treatments were fed to 25 Jersey cows (mean ± SD; 80 ± 14 d in milk) across 3 blocks in a partially balanced incomplete block design. Each block consisted of 4 periods of 28 d, where the final 4 d were used to determine milk production and composition, feed intake, energy utilization (via total collection and headbox-style indirect calorimetry), and N utilization (via total collection). Response surface models were used to evaluate treatment responses. Increasing dietary FA decreased DM intake and milk protein yield. When dietary starch was less than 24%, milk protein concentration increased with increasing sdLys, but when dietary starch was greater than 26% milk protein concentration decreased with increasing sdLys. Digestibility of FA increased when dietary FA increased from 3.0 to 4.2% and decreased as FA increased beyond 4.2%. Although neutral detergent fiber digestibility decreased as dietary starch increased, energy digestibility increased. As dietary FA increased, metabolizable energy (ME) content quadratically increased. Supply of ME increased as dietary FA increased from 3.0 to 4.2% and decreased as FA increased beyond 4.2%. Increasing dietary FA and starch decreased CH4 production and urinary energy. Increasing dietary starch increased the efficiency of utilizing dietary N for milk N. Increasing sdLys quadratically decreased N balance as sdLys increased from 0 to 8 g/d and increased N balance as sdLys increased from 8 to 18 g/d. Increasing dietary FA can increase ME content, however, at high dietary FA, decreased DM intake and FA digestibility resulted in a plateau in ME content and a decrease in ME supply. Our results demonstrate that sdLys supply is important for milk protein when dietary starch is low, and some Lys may be preferentially used for muscle protein synthesis at the expense of milk protein when sdLys is high.


Assuntos
Lactação , Amido , Animais , Bovinos , Dieta/veterinária , Digestão , Ácidos Graxos , Feminino , Lisina , Nitrogênio , Rúmen
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...