Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34.466
Filtrar
1.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 43(5): 696-705, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34728030

RESUMO

Objective To obtain the proteome and acetylome profiles of livers in mice during normal aging.Methods We applied tandem mass tag labeling and liquid chromatography tandem mass spectrometry and achieved proteome and acetylome data in C57BL/6J male mice aged 2 and 18 months under physiological conditions.Results A total of 4712 proteins were quantified by proteome profiling,and 4818 acetylated sites in 1367 proteins by acetylome profiling.The proteome and acetylome revealed moderate differences in the livers of young and old mice.There were 195 differentially expressed proteins in the proteome and 113 differentially expressed acetylated sites corresponding to 76 proteins in the acetylome.Functional enrichment analysis for the proteome showed that aging-associated upregulated proteins were mainly involved in fatty acid metabolism,epoxygenase P450 pathway,drug catabolic process,organic hydroxy compound metabolic process,and arachidonic acid metabolic process,while the downregulated proteins were related to regulation of gene silencing,nucleosome assembly,protein heterotetramerization,response to interferon,protein-DNA complex assembly and other processes.For the acetylome,the proteins with aging-associated upregulated acetylated sites mainly participated in cofactor metabolism,small molecule catabolic process,ribose phosphate metabolic process,ribonucleotide metabolic process,and purine-containing compound metabolic process,while the proteins with downregulated acetylated sites were associated with sulfur compound metabolic process,response to unfolded protein,and amino acid metabolic process.Conclusion We profiled the proteome and acetylome of livers in mice during normal aging and generated datasets for further research on aging.


Assuntos
Lisina , Proteoma , Acetilação , Envelhecimento , Animais , Fígado , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteoma/metabolismo
2.
Curr Protoc ; 1(11): e277, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34748287

RESUMO

Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.


Assuntos
Lisina Acetiltransferases , Lisina , Acetilação , Cromatografia Líquida , Lisina/metabolismo , Lisina Acetiltransferases/metabolismo , Espectrometria de Massas em Tandem
3.
BMC Genomics ; 22(1): 840, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34798813

RESUMO

BACKGROUND: Sanghuangporus sanghuang is a well-known traditional medicinal mushroom associated with mulberry. Despite the properties of this mushroom being known for many years, the regulatory mechanisms of bioactive compound biosynthesis in this medicinal mushroom are still unclear. Lysine malonylation is a posttranslational modification that has many critical functions in various aspects of cell metabolism. However, at present we do not know its role in S. sanghuang. In this study, a global investigation of the lysine malonylome in S. sanghuang was therefore carried out. RESULTS: In total, 714 malonyl modification sites were matched to 255 different proteins. The analysis indicated that malonyl modifications were involved in a wide range of cellular functions and displayed a distinct subcellular localization. Bioinformatics analysis indicated that malonylated proteins were engaged in different metabolic pathways, including glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Notably, a total of 26 enzymes related to triterpene and polysaccharide biosynthesis were found to be malonylated, indicating an indispensable role of lysine malonylation in bioactive compound biosynthesis in S. sanghuang. CONCLUSIONS: These findings suggest that malonylation is associated with many metabolic pathways, particularly the metabolism of the bioactive compounds triterpene and polysaccharide. This paper represents the first comprehensive survey of malonylation in S. sanghuang and provides important data for further study on the physiological function of lysine malonylation in S. sanghuang and other medicinal mushrooms.


Assuntos
Basidiomycota , Lisina , Basidiomycota/metabolismo , Biologia Computacional , Lisina/metabolismo , Processamento de Proteína Pós-Traducional
4.
Appl Microbiol Biotechnol ; 105(21-22): 8393-8410, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34617138

RESUMO

Feeding low-protein (LP) diets with essential amino acids could be an effective strategy for ruminants from economic, health and environmental perspectives. This study was conducted to investigate the effects of rumen-protected methionine and lysine (RML) in the LP diet on growth performance, innate immunity, and gut health of growing lambs. After 15 days of adaption, sixty-three male Hulunbuir lambs aged approximately 4 months were allotted to three dietary groups and each group had three pens with seven lambs for 60 days. The dietary treatments were as follows: a normal protein diet (14.5% CP, positive control; NP), LP diet (12.5% CP, negative control; LP), and LP diet with RML (12.5% CP, LP + RML). Lambs fed with LP + RML diet showed improved villus architecture and gut barrier function than those fed with the other two diets. The mRNA expressions of interleukin-1ß, tumor necrosis factor-α, interferon-γ, toll-like receptor-4, and myeloid differentiation primary response 88 were downregulated in most regions of the intestinal segments by feeding the LP + RML diet. Compared with the NP diet, feeding lambs with the LP diet increased the abundance of Candidatus_Saccharimonas in all regions of the intestinal tract and reversed by feeding the LP + RML diet. Lambs in the LP + RML diet group had lower abundance of Erysipelotrichaceae_UCG-009 and Clostridium_sensu_stricto_1 than those in the LP diet group. The results showed that supplementing RML in the LP diet exhibited beneficial effects on host immune function, intestinal mucosal integrity, and microbiota composition. KEY POINTS: • Adding methionine and lysine in a low-protein diet improve the intestinal mucosal growth and integrity. • Feeding a low-protein diet with methionine and lysine enhance the innate immune status. • Adding methionine and lysine in a low-protein diet alter the intestinal microbiota composition.


Assuntos
Dieta com Restrição de Proteínas , Microbioma Gastrointestinal , Ração Animal/análise , Animais , Lisina , Masculino , Metionina , Ovinos
5.
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
6.
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
7.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638563

RESUMO

BACKGROUND: Linoleic acid (LA) is an essential polyunsaturated fatty acid (PUFA) that is required for foetal growth and development. Excess intake of LA can be detrimental for metabolic health due to its pro-inflammatory properties; however, the effect of a diet high in LA on offspring metabolites is unknown. In this study, we aimed to determine the role of maternal or postnatal high linoleic acid (HLA) diet on plasma metabolites in adult offspring. METHODS: Female Wistar Kyoto (WKY) rats were fed with either low LA (LLA) or HLA diet for 10 weeks prior to conception and during gestation/lactation. Offspring were weaned at postnatal day 25 (PN25), treated with either LLA or HLA diets and sacrificed at PN180. Metabolite analysis was performed in plasma samples using Nuclear Magnetic Resonance. RESULTS: Maternal and postnatal HLA diet did not alter plasma metabolites in male and female adult offspring. There was no specific clustering among different treatment groups as demonstrated by principal component analysis. Interestingly, there was clustering among male and female offspring independent of maternal and postnatal dietary intervention. Lysine was higher in female offspring, while 3-hydroxybutyric acid and acetic acid were significantly higher in male offspring. CONCLUSION: In summary, maternal or postnatal HLA diet did not alter the plasma metabolites in the adult rat offspring; however, differences in metabolites between male and female offspring occurred independently of dietary intervention.


Assuntos
Ácido 3-Hidroxibutírico/sangue , Ácido Acético/sangue , Ácido Linoleico/administração & dosagem , Lisina/sangue , Crianças Adultas , Animais , Animais Recém-Nascidos , Dieta , Dieta Hiperlipídica , Feminino , Lactação , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Plasma/química , Plasma/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Análise de Componente Principal , Curva ROC , Ratos Endogâmicos WKY , Caracteres Sexuais
8.
BMC Bioinformatics ; 22(1): 519, 2021 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-34689734

RESUMO

BACKGROUND: Several computational tools for predicting protein Ubiquitylation and SUMOylation sites have been proposed to study their regulatory roles in gene location, gene expression, and genome replication. However, existing methods generally rely on feature engineering, and ignore the natural similarity between the two types of protein translational modification. This study is the first all-in-one deep network to predict protein Ubiquitylation and SUMOylation sites from protein sequences as well as their crosstalk sites simultaneously. Our deep learning architecture integrates several meta classifiers that apply deep neural networks to protein sequence information and physico-chemical properties, which were trained on multi-label classification mode for simultaneously identifying protein Ubiquitylation and SUMOylation as well as their crosstalk sites. RESULTS: The promising AUCs of our method on Ubiquitylation, SUMOylation and crosstalk sites achieved 0.838, 0.888, and 0.862 respectively on tenfold cross-validation. The corresponding APs reached 0.683, 0.804 and 0.552, which also validated our effectiveness. CONCLUSIONS: The proposed architecture managed to classify ubiquitylated and SUMOylated lysine residues along with their crosstalk sites, and outperformed other well-known Ubiquitylation and SUMOylation site prediction tools.


Assuntos
Aprendizado Profundo , Sumoilação , Sequência de Aminoácidos , Lisina/metabolismo , Ubiquitinação
9.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638970

RESUMO

Cardiovascular disease (CVD) is a common disease caused by many factors, including atherosclerosis, congenital heart disease, heart failure, and ischemic cardiomyopathy. CVD has been regarded as one of the most common diseases and has a severe impact on the life quality of patients. The main features of CVD include high morbidity and mortality, which seriously threaten human health. SUMO proteins covalently conjugate lysine residues with a large number of substrate proteins, and SUMOylation regulates the function of target proteins and participates in cellular activities. Under certain pathological conditions, SUMOylation of proteins related to cardiovascular development and function are greatly changed. Numerous studies have suggested that SUMOylation of substrates plays critical roles in normal cardiovascular development and function. We reviewed the research progress of SUMOylation in cardiovascular development and function, and the regulation of protein SUMOylation may be applied as a potential therapeutic strategy for CVD treatment.


Assuntos
Doenças Cardiovasculares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Animais , Doenças Cardiovasculares/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Coração/embriologia , Humanos , Lisina/metabolismo , Terapia de Alvo Molecular/métodos , Organogênese , Transdução de Sinais/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/metabolismo
10.
Nutrients ; 13(10)2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34684577

RESUMO

Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE-/-) mouse model. ApoE-/- mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay); (2) homocysteine by HPLC; (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS; and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio; (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI); and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p < 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.


Assuntos
Apolipoproteínas E/deficiência , Metilação de DNA/genética , Dieta Cetogênica , Epigênese Genética , Acetilação , Animais , Peso Corporal , Quimiocina CCL2/sangue , Histonas/metabolismo , Homocisteína/sangue , Cetose/sangue , Cetose/genética , Lisina/metabolismo , Masculino , Metaboloma , Camundongos , Projetos Piloto , Placa Aterosclerótica/sangue , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Processamento de Proteína Pós-Traducional
11.
Nat Struct Mol Biol ; 28(10): 811-824, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34608337

RESUMO

The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The inter-relatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics, we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.


Assuntos
Histonas/genética , Complexo Repressor Polycomb 1/genética , Iniciação da Transcrição Genética , Animais , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Histonas/metabolismo , Lisina/genética , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Polimerase II/metabolismo , Análise de Célula Única
12.
Nature ; 598(7879): 151-158, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34616067

RESUMO

The neocortex is disproportionately expanded in human compared with mouse1,2, both in its total volume relative to subcortical structures and in the proportion occupied by supragranular layers composed of neurons that selectively make connections within the neocortex and with other telencephalic structures. Single-cell transcriptomic analyses of human and mouse neocortex show an increased diversity of glutamatergic neuron types in supragranular layers in human neocortex and pronounced gradients as a function of cortical depth3. Here, to probe the functional and anatomical correlates of this transcriptomic diversity, we developed a robust platform combining patch clamp recording, biocytin staining and single-cell RNA-sequencing (Patch-seq) to examine neurosurgically resected human tissues. We demonstrate a strong correspondence between morphological, physiological and transcriptomic phenotypes of five human glutamatergic supragranular neuron types. These were enriched in but not restricted to layers, with one type varying continuously in all phenotypes across layers 2 and 3. The deep portion of layer 3 contained highly distinctive cell types, two of which express a neurofilament protein that labels long-range projection neurons in primates that are selectively depleted in Alzheimer's disease4,5. Together, these results demonstrate the explanatory power of transcriptomic cell-type classification, provide a structural underpinning for increased complexity of cortical function in humans, and implicate discrete transcriptomic neuron types as selectively vulnerable in disease.


Assuntos
Ácido Glutâmico/metabolismo , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neurônios/citologia , Neurônios/metabolismo , Doença de Alzheimer , Animais , Forma Celular , Colágeno/metabolismo , Eletrofisiologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Lisina/análogos & derivados , Masculino , Camundongos , Neocórtex/anatomia & histologia , Neurônios/classificação , Técnicas de Patch-Clamp , Transcriptoma
13.
PLoS One ; 16(10): e0256619, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34644302

RESUMO

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.


Assuntos
Ácidos Graxos/química , Mitocôndrias/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Sirtuína 3/metabolismo , Sirtuínas/metabolismo , Acetilação , Animais , Metabolismo Energético/fisiologia , Lipossomos/metabolismo , Fígado/metabolismo , Lisina/química , Camundongos , Camundongos Knockout , Mitocôndrias/enzimologia , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional , Sirtuína 3/genética , Sirtuínas/genética , Ácido Succínico/química
14.
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
15.
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
16.
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
17.
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
18.
Elife ; 102021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34524082

RESUMO

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.


Assuntos
Proteínas de Ciclo Celular , Heterocromatina , Código das Histonas/genética , Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Schizosaccharomyces pombe , Domínio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação/genética
19.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...