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1.
Proc Natl Acad Sci U S A ; 120(4): e2208941120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36656859

RESUMO

p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases , Metiltransferases , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Lisina/metabolismo , Metilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Proteína com Valosina/metabolismo , Metiltransferases/metabolismo
2.
Clin Sci (Lond) ; 137(1): 105-108, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36601782

RESUMO

Epigenome changes in chronic states of cardiovascular stress including diabetes, pressure overload and cardiomyopathies frequently involve changes in open chromatin and post-translation modifications of histone lysine residues at specific amino acid positions by acetylation, methylation and phosphorylation. Since the discovery of Set7 as an important regulator of histone H3 lysine 4 methylation state, there has been wide interest in its role in cardiovascular remodeling and cardiac dysfunction. Recent transcriptome and Fourier transform infrared spectroscopy analyses and in vivo assessments of cardiac function by Lunardon and colleagues now reveal a clear role of Set7 in the regulation of the extracellular matrix composition and cardiac hypertrophy in response to chronic isoproterenol induced cardiac stress.


Assuntos
Histona-Lisina N-Metiltransferase , Lisina , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Histonas/metabolismo , Cromatina , Metilação
3.
BMC Plant Biol ; 23(1): 23, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36631736

RESUMO

BACKGROUND: Protein lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM) discovered in cells or tissues of animals, microorganisms and plants in recent years. Proteome-wide identification of Khib-modified proteins has been performed in several plant species, suggesting that Khib-modified proteins are involved in a variety of biological processes and metabolic pathways. However, the protein Khib modification in soybean, a globally important legume crop that provides the rich source of plant protein and oil, remains unclear. RESULTS: In this study, the Khib-modified proteins in soybean leaves were identified for the first time using affinity enrichment and high-resolution mass spectrometry-based proteomic techniques, and a systematic bioinformatics analysis of these Khib-modified proteins was performed. Our results showed that a total of 4251 Khib sites in 1532 proteins were identified as overlapping in three replicates (the raw mass spectrometry data are available via ProteomeXchange with the identifier of PXD03650). These Khib-modified proteins are involved in a wide range of cellular processes, particularly enriched in biosynthesis, central carbon metabolism and photosynthesis, and are widely distributed in subcellular locations, mainly in chloroplasts, cytoplasm and nucleus. In addition, a total of 12 sequence motifs were extracted from all identified Khib peptides, and a basic amino acid residue (K), an acidic amino acid residue (E) and three aliphatic amino acid residues with small side chains (G/A/V) were found to be more preferred around the Khib site. Furthermore, 16 highly-connected clusters of Khib proteins were retrieved from the global PPI network, which suggest that Khib modifications tend to occur in proteins associated with specific functional clusters. CONCLUSIONS: These findings suggest that Khib modification is an abundant and conserved PTM in soybean and that this modification may play an important role in regulating physiological processes in soybean leaves. The Khib proteomic data obtained in this study will help to further elucidate the regulatory mechanisms of Khib modification in soybean in the future.


Assuntos
Haemophilus influenzae tipo b , Lisina , Animais , Lisina/metabolismo , Soja/genética , Soja/metabolismo , Haemophilus influenzae tipo b/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Processamento de Proteína Pós-Traducional
4.
Nucleus ; 14(1): 2160551, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36602897

RESUMO

Enhancers are cis-regulatory elements that can stimulate gene expression from distance, and drive precise spatiotemporal gene expression profiles during development. Functional enhancers display specific features including an open chromatin conformation, Histone H3 lysine 27 acetylation, Histone H3 lysine 4 mono-methylation enrichment, and enhancer RNAs production. These features are modified upon developmental cues which impacts their activity. In this review, we describe the current state of knowledge about enhancer functions and the diverse chromatin signatures found on enhancers. We also discuss the dynamic changes of enhancer chromatin signatures, and their impact on lineage specific gene expression profiles, during development or cellular differentiation.


Assuntos
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Lisina/metabolismo , Elementos Facilitadores Genéticos/genética , RNA
5.
Anal Biochem ; 663: 115032, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36592921

RESUMO

Protein 3-hydroxyl-3-methylglutarylation (HMGylation) is newly discovered lysine acylation modification in mitochondrion. The accurate identification of HMGylation sites is the premise and key to further explore the molecular mechanisms of HMGylation. In this study, a novel bioinformatics tool named HMGPred is developed to predict HMGylation sites. Multiple effective features, including amino acid composition, amino acid factors, binary encoding, and the composition of k-spaced amino acid pairs, are integrated to encode HMGylation sites. And F-score feature ranking with incremental feature selection was used to eliminate redundant features. Moreover, a fuzzy support vector machine algorithm is used to effectively reduce the influence of noise problem by assigning different samples to different fuzzy membership degrees. As illustrated by 10-fold cross-validation, HMGPred achieves a satisfactory performance with an area under receiver operating characteristic curve of 0.9110. Feature analysis indicates that some k-spaced amino acid pair features, such as 'KxxxT' and 'DxxxE', play a critical role in the prediction of HMGylation sites. The results of prediction and analysis might be helpful for investigating the mechanisms of HMGylation. For the convenience of experimental researchers, HMGPred is implemented as a web server at http://123.206.31.171/HMGPred/.


Assuntos
Lisina , Máquina de Vetores de Suporte , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/química , Aminoácidos/metabolismo , Algoritmos , Biologia Computacional/métodos
6.
Sci Rep ; 13(1): 377, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611042

RESUMO

Lysine methylation modulates the function of histone and non-histone proteins, and the enzymes that add or remove lysine methylation-lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively-are frequently mutated and dysregulated in human diseases. Identification of lysine methylation sites proteome-wide has been a critical barrier to identifying the non-histone substrates of KMTs and KDMs and for studying functions of non-histone lysine methylation. Detection of lysine methylation by mass spectrometry (MS) typically relies on the enrichment of methylated peptides by pan-methyllysine antibodies. In this study, we use peptide microarrays to show that pan-methyllysine antibodies have sequence bias, and we evaluate how the differential selectivity of these reagents impacts the detection of methylated peptides in MS-based workflows. We discovered that most commercially available pan-Kme antibodies have an in vitro sequence bias, and multiple enrichment approaches provide the most comprehensive coverage of the lysine methylome. Overall, global lysine methylation proteomics with multiple characterized pan-methyllysine antibodies resulted in the detection of 5089 lysine methylation sites on 2751 proteins from two human cell lines, nearly doubling the number of reported lysine methylation sites in the human proteome.


Assuntos
Lisina , Proteoma , Humanos , Lisina/metabolismo , Proteoma/metabolismo , Epigenoma , Metilação , Peptídeos/metabolismo , Anticorpos/metabolismo
7.
BMC Genomics ; 24(1): 23, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36647013

RESUMO

BACKGROUND: It is inevitable to change the function or expression of genes during the environmental adaption of species. Both the giant panda (Ailuropoda melanoleuca) and red panda (Ailurus fulgens) belong to Carnivora and have developed similar adaptations to the same dietary switch to bamboos at the morphological and genomic levels. However, the genetic adaptation at the gene expression level is unclear. Therefore, we aimed to examine the gene expression patterns of giant and red panda convergent specialized bamboo-diets. We examined differences in liver and pancreas transcriptomes between the two panda species and other non-herbivorous species. RESULTS: The clustering and PCA plots suggested that the specialized bamboo diet may drive similar expression shifts in these two species of pandas. Therefore, we focused on shared liver and pancreas DEGs (differentially expressed genes) in the giant and red panda relative to other non-herbivorous species. Genetic convergence occurred at multiple levels spanning carbohydrate metabolism, lipid metabolism, and lysine degradation. The shared adaptive convergence DEGs in both organs probably be an evolutionary response to the high carbohydrate, low lipid and lysine bamboo diet. Convergent expression of those nutrient metabolism-related genes in both pandas was an intricate process and subjected to multi-level regulation, including DNA methylation and transcription factor. A large number of lysine degradation and lipid metabolism related genes were hypermethylated in promoter regions in the red panda. Most genes related to carbohydrate metabolism had reduced DNA methylation with increased mRNA expression in giant pandas. Unlike the red panda, the core gene of the lysine degradation pathway (AASS) doesn't exhibit hypermethylation modification in the giant panda, and dual-luciferase reporter assay showed that transcription factor, NR3C1, functions as a transcriptional activator in AASS transcription through the binding to AASS promoter region. CONCLUSIONS: Our results revealed the adaptive expressions and regulations of the metabolism-related genes responding to the unique nutrients in bamboo food and provided data accumulation and research hints for the future revelation of complex mechanism of two pandas underlying convergent adaptation to a specialized bamboo diet.


Assuntos
Ailuridae , Dieta , Ursidae , Animais , Dieta/veterinária , Expressão Gênica , Lisina/metabolismo , Ursidae/genética , Ursidae/metabolismo , Ailuridae/genética , Ailuridae/metabolismo
8.
FASEB J ; 37(2): e22747, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36607631

RESUMO

SOHLH1 and NOBOX are oocyte-expressed transcription factors with critical roles in ovary development and fertility. In mice, Sohlh1 and Nobox are essential for fertility through their regulation of the oocyte transcriptional network and cross-talk to somatic cells. Sumoylation is a posttranslational modification that regulates transcription factor function, and we previously showed that mouse oocytes deficient for sumoylation had an altered transcriptional landscape that included significant changes in NOBOX target genes. Here, we show that mouse SOHLH1 is modified by SUMO2/3 at lysine 345 and mutation of this residue alters SOHLH1 nuclear to cytoplasmic localization. In NOBOX, we identify a non-consensus SUMO site, K97, that eliminates NOBOX mono-SUMO2/3 conjugation, while a point mutation at K125 had no effect on NOBOX sumoylation. However, NOBOXK97R/K125R double mutants showed loss of mono-SUMO2/3 and altered higher molecular weight modifications, suggesting cooperation between these lysine's. NOBOXK97R and NOBOXK97R/K125R differentially regulated NOBOX promoter targets, with increased activity on the Gdf9 promoter, but no effect on the Pou5f1 promoter. These data implicate sumoylation as a novel regulatory mechanism for SOHLH1 and NOBOX, which may prove useful in refining their roles during oogenesis as well as their function during reprogramming to generate de novo germ cells.


Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Animais , Feminino , Camundongos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/genética , Lisina/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo
9.
Cell Mol Life Sci ; 80(2): 47, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36658409

RESUMO

Histone H3 trimethylation on lysine 9 (H3K9me3) is a defining feature of mammalian pericentromeres, loss of which results in genome instability. Here we show that CDYL2 is recruited to pericentromeres in an H3K9me3-dependent manner and is required for faithful mitosis and genome stability. CDYL2 RNAi in MCF-7 breast cancer cells and Hela cervical cancer cells inhibited their growth, induced apoptosis, and provoked both nuclear and mitotic aberrations. Mass spectrometry analysis of CDYL2-interacting proteins identified the neurodevelopmental disease-linked mitotic regulators CHAMP1 and POGZ, which are associated with a central non-conserved region of CDYL2. RNAi rescue assays identified both the CDYL2 chromodomain and the CHAMP1-POGZ interacting region as required and, together, sufficient for CDYL2 regulation of mitosis and genome stability. CDYL2 RNAi caused loss of CHAMP1 localization at pericentromeres. We propose that CDYL2 functions as an adaptor protein that connects pericentromeric H3K9me3 with CHAMP1 and POGZ to ensure mitotic fidelity and genome stability.


Assuntos
Proteínas Cromossômicas não Histona , Proteínas Correpressoras , Histonas , Mitose , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Instabilidade Genômica , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Mitose/genética , Fosfoproteínas/metabolismo , Interferência de RNA , Células MCF-7 , Proteínas Correpressoras/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo
10.
Cells ; 12(2)2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36672228

RESUMO

Seed germination is the first step in initiating a new life cycle in seed plants. Light is a major environmental factor affecting seed germination. Phytochrome B (phyB) is the primary photoreceptor promoting germination during the initial phase of imbibition. Post-translational histone methylation occurring at both lysine and arginine residues plays a crucial role in transcriptional regulation in plants. However, the role of histone lysine demethylation in light-initiated seed germination is not yet reported. Here, we identified that Relative of Early Flowering 6 (REF6)/Jumonji Domain-containing Protein 12 (JMJ12), a histone H3 lysine 27 (H3K27) demethylase, acts as a positive regulator of light-initiated seed germination. The loss of function of REF6 in Arabidopsis inhibits phyB-dependent seed germination. Genome-wide RNA-sequencing analysis revealed that REF6 regulates about half of the light-responsive transcriptome in imbibed seeds, including genes related to multiple hormonal signaling pathways and cellular processes. Phenotypic analyses indicated that REF6 not only regulates seed germination through GA (gibberellin) and ABA (abscisic acid) processes but also depends on the auxin signaling pathway. Furthermore, REF6 directly binds to and decreases the histone H3K27me3 levels of auxin-signaling- and cell-wall-loosening-related genes, leading to the activated expression of these genes in imbibed seeds. Taken together, our study identifies REF6 as the first histone lysine demethylase required for light-initiated seed germination. Our work also reveals the important role of REF6-mediated histone H3K27 demethylation in transcriptional reprogramming in the light-initiated seed germination process.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Germinação/genética , Histonas/metabolismo , Proteínas de Arabidopsis/metabolismo , Histona Desmetilases/metabolismo , Lisina/metabolismo , Sementes/genética , Sementes/metabolismo , Ácidos Indolacéticos/metabolismo , Fatores de Transcrição/metabolismo
11.
Genes (Basel) ; 14(1)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36672956

RESUMO

Histone lysine methyltransferase and demethylase enzymes play a central role in chromatin organization and gene expression through the dynamic regulation of histone lysine methylation. Consistent with this, genes encoding for histone lysine methyltransferases (KMTs) and demethylases (KDMs) are involved in complex human syndromes, termed congenital regulopathies. In this report, we present several lines of evidence for the involvement of these genes in developmental ocular phenotypes, suggesting that individuals with structural eye defects, especially when accompanied by craniofacial, neurodevelopmental and growth abnormalities, should be examined for possible variants in these genes. We identified nine heterozygous damaging genetic variants in KMT2D (5) and four other histone lysine methyltransferases/demethylases (KMT2C, SETD1A/KMT2F, KDM6A and KDM5C) in unrelated families affected with developmental eye disease, such as Peters anomaly, sclerocornea, Axenfeld-Rieger spectrum, microphthalmia and coloboma. Two families were clinically diagnosed with Axenfeld-Rieger syndrome and two were diagnosed with Peters plus-like syndrome; others received no specific diagnosis prior to genetic testing. All nine alleles were novel and five of them occurred de novo; five variants resulted in premature truncation, three were missense changes and one was an in-frame deletion/insertion; and seven variants were categorized as pathogenic or likely pathogenic and two were variants of uncertain significance. This study expands the phenotypic spectra associated with KMT and KDM factors and highlights the importance of genetic testing for correct clinical diagnosis.


Assuntos
Anormalidades do Olho , Histonas , Humanos , Histonas/genética , Lisina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Anormalidades do Olho/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo
12.
Biotechnol Bioeng ; 120(1): 312-317, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36226358

RESUMO

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.


Assuntos
Ácido 2-Aminoadípico , Lisina , Ácido 2-Aminoadípico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lisina/metabolismo , Saccharomyces cerevisiae/metabolismo , Sacaropina Desidrogenases/metabolismo
13.
Methods Mol Biol ; 2589: 411-428, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255640

RESUMO

Protein lysine acylation represents one of the most common post-translational modifications. Obviously, highly reactive metabolic intermediates, like thioesters and mixed anhydrides between phosphoric acid and organic acids, modify lysine residues spontaneously. Additionally, enzymes using acyl-CoAs as co-substrates transfer the acyl residue specifically to defined sequences within proteins. The counteracting enzymes are called histone deacetylases (HDACs), releasing the free lysine side chain. Such enzymatic activities are involved in different cellular processes like tumor progression, immune response, regulation of metabolism, and aging. Modulators of such enzymatic activities represent valuable tools in drug discovery. Therefore, direct and continuous assays to monitor enzymatic activity of HDACs are needed. Here we describe different assay formats allowing both monitoring of Zn2+-dependent HDACs via UV-Vis-spectroscopy and NAD+-dependent HDACs (sirtuins) by fluorescence-based assay formats. Additionally, we describe methods enabling efficient screening of HDAC-inhibitors via fluorescence displacement assays.


Assuntos
Histonas , Sirtuínas , Lisina/metabolismo , NAD/metabolismo , Histona Desacetilases/metabolismo , Sirtuínas/metabolismo , Ácidos Fosfóricos/metabolismo , Anidridos
14.
Biochim Biophys Acta Biomembr ; 1865(2): 184098, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36481181

RESUMO

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.


Assuntos
Apolipoproteína A-I , Lisina , Apolipoproteína A-I/genética , Apolipoproteína A-I/química , Ligação Proteica , Lisina/genética , Lisina/metabolismo , Glutamina/metabolismo , Dicroísmo Circular
15.
EBioMedicine ; 87: 104418, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36584593

RESUMO

Metabolism regulates cardiovascular biology through multiple mechanisms, including epigenetic modifications. Over the past two decades, experimental and preclinical studies have highlighted the critical roles of histone modifications in cardiovascular development, homeostasis, and diseases. The widely studied histone acetylation is critical in cardiovascular biology and diseases, and inhibitors of histone deacetylases show therapeutic values. In addition to lysine acetylation, a series of novel non-acetyl lysine acylations have recently been recognized. These non-acetyl lysine acylations have been demonstrated to have physiological and pathological functions, and recent studies have analyzed the roles of these non-acetyl lysine acylations in cardiovascular biology. Herein, we review the current advances in the understanding of non-acetyl lysine acylations in cardiovascular biology and discuss open questions and translational perspectives. These new pieces of evidence provide a more extensive insight into the epigenetic mechanisms underlying cardiovascular biology and help assess the feasibility of targeting acylations to treat cardiovascular diseases.


Assuntos
Histonas , Lisina , Humanos , Acetilação , Histonas/metabolismo , Lisina/metabolismo , Acilação , Processamento de Proteína Pós-Traducional , Biologia
16.
Biogerontology ; 24(1): 137-148, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36550376

RESUMO

Aging-affected cellular compositions of the spinal cord are diverse and region specific. Age leads to the accumulation of abnormal protein aggregates and dysregulation of proteostasis. Dysregulated proteostasis and protein aggregates result from dysfunction of the ubiquitin-proteasome system (UPS) and autophagy. Understanding the molecular mechanisms of spinal cord aging is essential and important for scientists to discover new therapies for rejuvenation. We found age-related increases in STAT3 and decreases in Tuj1 in aging mouse spinal cords, which was characterized by increased expression of P16. Coaggregation of lysine-48 and lysine-63 ubiquitin with STAT3 was revealed in aging mouse spinal cords. STAT3-ubiquitin aggregates formed via lysine-48 and lysine-63 linkages were increased significantly in the aging spinal cords but not in central canal ependymal cells or neural stem cells in the spinal cord. These results highlight the increase in STAT3 and its region-specific aggregation and ubiquitin-conjugation during spinal cord aging.


Assuntos
Envelhecimento , Células-Tronco Neurais , Fator de Transcrição STAT3 , Animais , Masculino , Camundongos , Envelhecimento/metabolismo , Lisina/metabolismo , Células-Tronco Neurais/metabolismo , Agregados Proteicos , Medula Espinal/metabolismo , Fator de Transcrição STAT3/metabolismo , Ubiquitinas/metabolismo
17.
Mol Metab ; 67: 101653, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36513219

RESUMO

BACKGROUND: Key cellular metabolites reflecting the immediate activity of metabolic enzymes as well as the functional metabolic state of intracellular organelles can act as powerful signal regulators to ensure the activation of homeostatic responses. The citrate/acetyl-CoA pathway, initially recognized for its role in intermediate metabolism, has emerged as a fundamental branch of this nutrient-sensing homeostatic response. Emerging studies indicate that fluctuations in acetyl-CoA availability within different cellular organelles and compartments provides substrate-level regulation of many biological functions. A fundamental aspect of these regulatory functions involves Nε-lysine acetylation. SCOPE OF REVIEW: Here, we will examine the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis. These functions will be analyzed in the context of associated human diseases and specific mouse models of dysfunctional ER acetylation and citrate/acetyl-CoA flux. A primary objective of this review is to highlight the complex yet integrated response of compartment- and organelle-specific Nε-lysine acetylation to the intracellular availability and flux of acetyl-CoA, linking this important post-translational modification to cellular metabolism. MAJOR CONCLUSIONS: The ER acetylation machinery regulates the proteostatic functions of the organelle as well as the metabolic crosstalk between different intracellular organelles and compartments. This crosstalk enables the cell to impart adaptive responses within the ER and the secretory pathway. However, it also enables the ER to impart adaptive responses within different cellular organelles and compartments. Defects in the homeostatic balance of acetyl-CoA flux and ER acetylation reflect different but converging disease states in humans as well as converging phenotypes in relevant mouse models. In conclusion, citrate and acetyl-CoA should not only be seen as metabolic substrates of intermediate metabolism but also as signaling molecules that direct functional adaptation of the cell to both intracellular and extracellular messages. Future discoveries in CoA biology and acetylation are likely to yield novel therapeutic approaches.


Assuntos
Ácido Cítrico , Lisina , Camundongos , Animais , Humanos , Acetilcoenzima A/metabolismo , Lisina/metabolismo , Ácido Cítrico/metabolismo , Acetilação , Processamento de Proteína Pós-Traducional , Retículo Endoplasmático/metabolismo , Citratos/metabolismo
18.
Microbiol Res ; 268: 127296, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36580869

RESUMO

The protein lysine acetylation includes acetyl-CoA (AcCoA) or acetyl phosphate (AcP)-mediated nonenzymatic acetylation, and enzymatic acetylation. It is widespread in the proteomes but the acetylation levels of most sites are very low. A thorough understanding of the determinants of low acetylation levels is highly important for elucidating the physiological relevance of lysine acetylation. In this study, we constructed a non-native substrate library containing 24 synthesized polypeptides, and we showed that ATP could inhibit the AcCoA-mediated nonenzymatic acetylation of these polypeptides through LC-MS/MS analysis. The acetyltransferase PatZ could acetylated these non-native substrates, and the PatZ-catalyzed acetylation of the polypeptides was also inhibited by ATP. Furthermore, the Western blot showed that ATP also inhibited the nonenzymatic (AcCoA or AcP-mediated) and enzymatic (PatZ-catalyzed) acetylation of acetyl-CoA synthetase Acs, which is a native substrate for acetylation. ATP can also inhibit the autoacetylation of acetyltransferase PatZ. Besides, both ADP and AMP could enhance the AcP-mediated acetylation of Acs, but ADP slightly inhibited the AcCoA-mediated acetylation of Acs. However, both ADP and AMP had no evident inhibition on the PatZ-catalyzed acetylation of Acs. Based on these results, we proposed that ATP can act as an inhibitor of acetylation, and it may regulate the function of PatZ by inhibiting its autoacetylation and compensate for the function of deacetylase CobB.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Trifosfato de Adenosina/metabolismo , Lisina/metabolismo , Acetilação , Acetilcoenzima A/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Acetiltransferases , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo
19.
Redox Biol ; 59: 102574, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36521306

RESUMO

Mice with ectopic expression of uncoupling protein-1 (UCP1) in skeletal muscle exhibit a healthy aging phenotype with increased longevity and resistance to impaired metabolic health. This may be achieved by decreasing protein glycation by the reactive metabolite, methylglyoxal (MG). We investigated protein glycation and oxidative damage in skeletal muscle of mice with UCP1 expression under control of the human skeletal actin promoter (HSA-mUCP1) at age 12 weeks (young) and 70 weeks (aged). We found both young and aged HSA-mUCP1 mice had decreased advanced glycation endproducts (AGEs) formed from MG, lysine-derived Nε(1-carboxyethyl)lysine (CEL) and arginine-derived hydroimidazolone, MG-H1, whereas protein glycation by glucose forming Nε-fructosyl-lysine (FL) was increased ca. 2-fold, compared to wildtype controls. There were related increases in FL-linked AGEs, Nε-carboxymethyl-lysine (CML) and 3-deoxylglucosone-derived hydroimidazolone 3DG-H, and minor changes in protein oxidative and nitration adducts. In aged HSA-mUCP1 mice, urinary MG-derived AGEs/FL ratio was decreased ca. 60% whereas there was no change in CML/FL ratio - a marker of oxidative damage. This suggests that, normalized for glycemic status, aged HSA-mUCP1 mice had a lower flux of whole body MG-derived AGE exposure compared to wildtype controls. Proteomics analysis of skeletal muscle revealed a shift to increased heat shock proteins and mechanoprotection and repair in HSA-mUCP1 mice. Decreased MG-derived AGE protein content in skeletal muscle of aged HSA-mUCP1 mice is therefore likely produced by increased proteolysis of MG-modified proteins and increased proteostasis surveillance of the skeletal muscle proteome. From this and previous transcriptomic studies, signaling involved in enhanced removal of MG-modified protein is likely increased HSPB1-directed HUWE1 ubiquitination through eIF2α-mediated, ATF5-induced increased expression of HSPB1. Decreased whole body exposure to MG-derived AGEs may be linked to increased weight specific physical activity of HSA-mUCP1 mice. Decreased formation and increased clearance of MG-derived AGEs may be associated with healthy aging in the HSA-mUCP1 mouse.


Assuntos
Produtos Finais de Glicação Avançada , Envelhecimento Saudável , Humanos , Camundongos , Animais , Idoso , Lactente , Produtos Finais de Glicação Avançada/metabolismo , Lisina/metabolismo , Aldeído Pirúvico/metabolismo , Reação de Maillard , Proteína Desacopladora 1/metabolismo , Expressão Ectópica do Gene , Proteínas/metabolismo , Músculo Esquelético/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
20.
J Proteomics ; 250: 104383, 2022 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-34562664

RESUMO

Lysine 2-hydroxyisobutyrylation (Khib) is one of the newly discovered post-translational modifications (PTMs) through protein acylation. It has been reported to be widely distributed in both eukaryotes and prokaryotes, and plays an important role in chromatin conformation change, gene transcription, protein subcellular localization, protein-protein interaction, signal transduction, and cellular proliferation. In this study, the Khib modification proteome of siliques from A. thaliana under salt stress (Ss) and those in the control (Cs) were compared. The results showed that Khib modification was abundant in siliques. Totally 3810 normalized Khib sites on 1254 proteins were identified, and the Khib modification showed a downregulation trend dramatically: it was down-regulated at 282 sites on 205 proteins while was up-regulated at 96 sites on 78 proteins in Ss siliques (Data are available via ProteomeXchange with identifier PXD028116 and PXD026643). Among them, 13 proteins, including F4IVN6, Q9M1P5, and Q9LF33, had sites with the most significant regulation of Khib modification. Bioinformatics analysis suggested that the differentially Khib-regulated proteins mainly participated in glycolysis/gluconeogenesis and endocytosis. In particular, there were differentially117 Khib-regulated proteins that were mapped to the protein-protein interaction database. In the KEGG pathway enrichment analysis, Khib-modified proteins were enriched in several pathways related to energy metabolism, including gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism. Overall, our work reveals the first systematic analysis of Khib proteome in Arabidopsis siliques under salt stress, and sheds a light on the future studies on the regulatory mechanisms of Khib during the salt stress response of plants. SIGNIFICANCE: In this study, we found the Khib-modified proteins in silique under salt stress and described the enrichment of Khib-modified proteins involved in the biological processes and cellular localization. Proteins undergoing 2-hydroxyisobutylation were mainly involved in the gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism, suggesting that 2-hydroxyisobutylation affects the energy metabolic pathway, and thus the development of the plant. In addition, specific candidate proteins that may affect plant development under salt stress were selected. This study will provide a theoretical basis for revealing the function and mechanism of these proteins and their 2-hydroxyisobutyryl modifications during the development of silique under salt stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Estresse Salino
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