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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Nat Commun ; 12(1): 5098, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429416

RESUMO

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons/fisiologia , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Fenilalanina , Potássio/metabolismo , Subunidades Proteicas , ATPase Trocadora de Sódio-Potássio
9.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360722

RESUMO

Biological aging research is expected to reveal modifiable molecular mechanisms that can be harnessed to slow or possibly reverse unhealthy trajectories. However, there is first an urgent need to define consensus molecular markers of healthy and unhealthy aging. Established aging hallmarks are all linked to metabolism, and a 'rewired' metabolic circuitry has been shown to accelerate or delay biological aging. To identify metabolic signatures distinguishing healthy from unhealthy aging trajectories, we performed nontargeted metabolomics on skeletal muscles from 2-month-old and 21-month-old mice, and after dietary and lifestyle interventions known to impact biological aging. We hypothesized that common metabolic signatures would highlight specific pathways and processes promoting healthy aging, while revealing the molecular underpinnings of unhealthy aging. Here, we report 50 metabolites that commonly distinguished aging trajectories in all cohorts, including 18 commonly reduced under unhealthy aging and 32 increased. We stratified these metabolites according to known relationships with various aging hallmarks and found the greatest associations with oxidative stress and nutrient sensing. Collectively, our data suggest interventions aimed at maintaining skeletal muscle arginine and lysine may be useful therapeutic strategies to minimize biological aging and maintain skeletal muscle health, function, and regenerative capacity in old age.


Assuntos
Envelhecimento/metabolismo , Arginina/metabolismo , Lisina/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Transdução de Sinais , Envelhecimento/patologia , Animais , Masculino , Camundongos , Músculo Esquelético/patologia
10.
Int J Mol Sci ; 22(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34445236

RESUMO

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.


Assuntos
Proteínas de Transporte/metabolismo , Lipopolissacarídeos/toxicidade , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Sepse/enzimologia , Hormônios Tireóideos/metabolismo , Acetilação/efeitos dos fármacos , Células Hep G2 , Humanos , Lisina/metabolismo , Sepse/induzido quimicamente
11.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445771

RESUMO

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.


Assuntos
Corynebacterium glutamicum/metabolismo , Ácido Diaminopimélico/metabolismo , Lisina/metabolismo , Transdução de Sinais/fisiologia , Aciltransferases/metabolismo , Compostos de Amônio/metabolismo , Oxirredutases/metabolismo
12.
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
13.
EMBO Rep ; 22(10): e52023, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34369651

RESUMO

Histone lysine crotonylation (Kcr), an evolutionarily conserved and widespread non-acetyl short-chain lysine acylation, plays important roles in transcriptional regulation and disease processes. However, the genome-wide distribution, dynamic changes, and associations with gene expression of histone Kcr during developmental processes are largely unknown. In this study, we find that histone Kcr is mainly located in active promoter regions, acts as an epigenetic hallmark of highly expressed genes, and regulates genes participating in metabolism and proliferation. Moreover, elevated histone Kcr activates bivalent promoters to stimulate gene expression in neural stem/progenitor cells (NSPCs) by increasing chromatin openness and recruitment of RNA polymerase II (RNAP2). Functionally, these activated genes contribute to transcriptome remodeling and promote neuronal differentiation. Overall, histone Kcr marks active promoters with high gene expression and modifies the local chromatin environment to allow gene activation.


Assuntos
Histonas , Células-Tronco Neurais , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Células-Tronco Neurais/metabolismo , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional
14.
Cancer Sci ; 112(10): 3935-3944, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34251718

RESUMO

Homeostasis of the hematopoietic system is achieved in a hierarchy, with hematopoietic stem cells at the pinnacle. Because only hematopoietic stem cells (HSCs) can self-renew, the size of the hematopoietic system is strictly controlled. In hematopoietic reconstitution experiments, 1 HSC can reconstitute the entire hematopoietic system, whereas 50 multipotent progenitors cannot. This indicates that only HSCs self-renew, whereas non-HSC hematopoietic progenitors are programmed to differentiate or senesce. Oncogenic mutations of the mixed lineage leukemia gene (MLL) overcome this "programmed differentiation" by conferring the self-renewing ability to non-HSC hematopoietic progenitors. In leukemia, mutated MLL proteins constitutively activate a broad range of previously transcribed CpG-rich promoters by an MLL-mediated transcriptional activation system. This system promotes self-renewal by replicating an expression profile similar to that of the mother cell in its daughter cells. In this transcriptional activation system, MLL binds to unmethylated CpG-rich promoters and recruits RNA polymerase II. MLL recruits p300/CBP through its transcriptional activation domain, which acetylates histone H3 at lysines 9, 18, and 27. The AF4 family/ENL family/P-TEFb complex (AEP) binds to acetylated H3K9/18/27 to activate transcription. Gene rearrangements of MLL with AEP- or CBP/p300-complex components generate constitutively active transcriptional machinery of this transcriptional activation system, which causes aberrant self-renewal of leukemia stem cells. Inhibitors of the components of this system effectively decrease their leukemogenic potential.


Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Histona-Lisina N-Metiltransferase/genética , Leucemia/etiologia , Proteína de Leucina Linfoide-Mieloide/genética , Ativação Transcricional/fisiologia , Acetilação , Diferenciação Celular , Autorrenovação Celular/genética , Senescência Celular , Ilhas de CpG/genética , Proteínas de Ligação a DNA/metabolismo , Proteína p300 Associada a E1A/metabolismo , Rearranjo Gênico , Hematopoese/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Leucemia/prevenção & controle , Lisina/metabolismo , Células-Tronco Multipotentes/fisiologia , Mutação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/metabolismo
15.
Nature ; 596(7872): 433-437, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321663

RESUMO

Protein ubiquitination at sites of DNA double-strand breaks (DSBs) by RNF168 recruits BRCA1 and 53BP11,2, which are mediators of the homologous recombination and non-homologous end joining DSB repair pathways, respectively3. Non-homologous end joining relies on 53BP1 binding directly to ubiquitinated lysine 15 on H2A-type histones (H2AK15ub)4,5 (which is an RNF168-dependent modification6), but how RNF168 promotes BRCA1 recruitment and function remains unclear. Here we identify a tandem BRCT-domain-associated ubiquitin-dependent recruitment motif (BUDR) in BRCA1-associated RING domain protein 1 (BARD1) (the obligate partner protein of BRCA1) that, by engaging H2AK15ub, recruits BRCA1 to DSBs. Disruption of the BUDR of BARD1 compromises homologous recombination and renders cells hypersensitive to PARP inhibition and cisplatin. We further show that BARD1 binds nucleosomes through multivalent interactions: coordinated binding of H2AK15ub and unmethylated H4 lysine 20 by its adjacent BUDR and ankyrin repeat domains, respectively, provides high-affinity recognition of DNA lesions in replicated chromatin and promotes the homologous recombination activities of the BRCA1-BARD1 complex. Finally, our genetic epistasis experiments confirm that the need for BARD1 chromatin-binding activities can be entirely relieved upon deletion of RNF168 or 53BP1. Thus, our results demonstrate that by sensing DNA-damage-dependent and post-replication histone post-translation modification states, BRCA1-BARD1 complexes coordinate the antagonization of the 53BP1 pathway with promotion of homologous recombination, establishing a simple paradigm for the governance of the choice of DSB repair pathway.


Assuntos
Recombinação Homóloga , Lisina/química , Lisina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Adulto , Motivos de Aminoácidos , Proteína BRCA1/química , Proteína BRCA1/metabolismo , Cromatina/metabolismo , Cisplatino/farmacologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos dos fármacos , Feminino , Células HCT116 , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Domínios Proteicos , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/deficiência
16.
Mol Cell ; 81(18): 3833-3847.e11, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34289383

RESUMO

Mutant isocitrate dehydrogenase (IDH) 1 and 2 play a pathogenic role in cancers, including acute myeloid leukemia (AML), by producing oncometabolite 2-hydroxyglutarate (2-HG). We recently reported that tyrosine phosphorylation activates IDH1 R132H mutant in AML cells. Here, we show that mutant IDH2 (mIDH2) R140Q commonly has K413 acetylation, which negatively regulates mIDH2 activity in human AML cells by attenuating dimerization and blocking binding of substrate (α-ketoglutarate) and cofactor (NADPH). Mechanistically, K413 acetylation of mitochondrial mIDH2 is achieved through a series of hierarchical phosphorylation events mediated by tyrosine kinase FLT3, which phosphorylates mIDH2 to recruit upstream mitochondrial acetyltransferase ACAT1 and simultaneously activates ACAT1 and inhibits upstream mitochondrial deacetylase SIRT3 through tyrosine phosphorylation. Moreover, we found that the intrinsic enzyme activity of mIDH2 is much higher than mIDH1, thus the inhibitory K413 acetylation optimizes leukemogenic ability of mIDH2 in AML cells by both producing sufficient 2-HG for transformation and avoiding cytotoxic accumulation of intracellular 2-HG.


Assuntos
Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Acetilação , Animais , Antineoplásicos/farmacologia , Feminino , Humanos , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Mutação/genética , NADP/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Polimorfismo de Nucleotídeo Único/genética , Cultura Primária de Células , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo
17.
Nat Genet ; 53(8): 1221-1232, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34294917

RESUMO

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.


Assuntos
Elementos Facilitadores Genéticos , Histonas/metabolismo , Células-Tronco Neurais/fisiologia , Complexo Repressor Polycomb 2/genética , Rombencéfalo/citologia , Animais , Neoplasias Encefálicas/genética , Diferenciação Celular/genética , Linhagem Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Epigenoma , Regulação da Expressão Gênica no Desenvolvimento , Glioma/genética , Histonas/genética , Humanos , Lisina/metabolismo , Masculino , Camundongos Endogâmicos , Mutação , Células-Tronco Neurais/transplante , Oncogenes , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Rombencéfalo/fisiologia
18.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208020

RESUMO

Herpes simplex virus 1 (HSV-1) is a widespread neurotropic virus establishing a life-long latent infection in neurons with periodic reactivations. Recent studies linked HSV-1 to neurodegenerative processes related to age-related disorders such as Alzheimer's disease. Here, we explored whether recurrent HSV-1 infection might accelerate aging in neurons, focusing on peculiar marks of aged cells, such as the increase in histone H4 lysine (K) 16 acetylation (ac) (H4K16ac); the decrease of H3K56ac, and the modified expression of Sin3/HDAC1 and HIRA proteins. By exploiting both in vitro and in vivo models of recurrent HSV-1 infection, we found a significant increase in H4K16ac, Sin3, and HDAC1 levels, suggesting that the neuronal response to virus latency and reactivation includes the upregulation of these aging markers. On the contrary, we found a significant decrease in H3K56ac that was specifically linked to viral reactivation and apparently not related to aging-related markers. A complex modulation of HIRA expression and localization was found in the brain from HSV-1 infected mice suggesting a specific role of this protein in viral latency and reactivation. Overall, our results pointed out novel molecular mechanisms through which recurrent HSV-1 infection may affect neuronal aging, likely contributing to neurodegeneration.


Assuntos
Senescência Celular , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Modelos Biológicos , Neurônios/patologia , Neurônios/virologia , Acetilação , Animais , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Chaperonas de Histonas/metabolismo , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Ratos Wistar , Recidiva , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Fatores de Transcrição/metabolismo , Latência Viral
19.
BMC Genomics ; 22(1): 542, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34266380

RESUMO

BACKGROUND: Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered protein posttranslational modification (PTM) and is involved in the broad-spectrum regulation of cellular processes that are found in both prokaryotic and eukaryotic cells, including in plants. The Chinese herb rhubarb (Dahuang) is one of the most widely used traditional Chinese medicines in clinical applications. To better understand the physiological activities and mechanism of treating diseases with the herb, it is necessary to conduct intensive research on rhubarb. However, Khib modification has not been reported thus far in rhubarb. RESULTS: In this study, we performed the first global analysis of Khib-modified proteins in rhubarb by using sensitive affinity enrichment combined with high-accuracy HPLC-MS/MS tandem spectrometry. A total of 4333 overlapping Khib modification peptides matched on 1525 Khib-containing proteins were identified in three independent tests. Bioinformatics analysis showed that these Khib-containing proteins are involved in a wide range of cellular processes, particularly in protein biosynthesis and central carbon metabolism and are distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria. In addition, the amino acid sequence motif analysis showed that a negatively charged side chain residue (E), a positively charged residue (K), and an uncharged residue with the smallest side chain (G) were strongly preferred around the Khib site, and a total of 13 Khib modification motifs were identified. These identified motifs can be classified into three motif patterns, and some motif patterns are unique to rhubarb and have not been identified in other plants to date. CONCLUSIONS: A total of 4333 Khib-modified peptides on 1525 proteins were identified. The Khib-modified proteins are mainly distributed in the chloroplast, cytoplasm, nucleus and mitochondria, and involved in a wide range of cellular processes. Moreover, three types of amino acid sequence motif patterns, including EKhib/KhibE, GKhib and k.kkk….Khib….kkkkk, were extracted from a total of 13 Khib-modified peptides. This study provides comprehensive Khib-proteome resource of rhubarb. The findings from the study contribute to a better understanding of the physiological roles of Khib modification, and the Khib proteome data will facilitate further investigations of the roles and mechanisms of Khib modification in rhubarb.


Assuntos
Haemophilus influenzae tipo b , Rheum , China , Haemophilus influenzae tipo b/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Rheum/metabolismo , Espectrometria de Massas em Tandem
20.
Nat Commun ; 12(1): 4482, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301959

RESUMO

Activation of Pannexin 1 (PANX1) ion channels causes release of intercellular signaling molecules in a variety of (patho)physiological contexts. PANX1 can be activated by G protein-coupled receptors (GPCRs), including α1-adrenergic receptors (α1-ARs), but how receptor engagement leads to channel opening remains unclear. Here, we show that GPCR-mediated PANX1 activation can occur via channel deacetylation. We find that α1-AR-mediated activation of PANX1 channels requires Gαq but is independent of phospholipase C or intracellular calcium. Instead, α1-AR-mediated PANX1 activation involves RhoA, mammalian diaphanous (mDia)-related formin, and a cytosolic lysine deacetylase activated by mDia - histone deacetylase 6. HDAC6 associates with PANX1 and activates PANX1 channels, even in excised membrane patches, suggesting direct deacetylation of PANX1. Substitution of basally-acetylated intracellular lysine residues identified on PANX1 by mass spectrometry either prevents HDAC6-mediated activation (K140/409Q) or renders the channels constitutively active (K140R). These data define a non-canonical RhoA-mDia-HDAC6 signaling pathway for GαqPCR activation of PANX1 channels and uncover lysine acetylation-deacetylation as an ion channel silencing-activation mechanism.


Assuntos
Conexinas/metabolismo , Desacetilase 6 de Histona/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetilação , Animais , Células Cultivadas , Conexinas/genética , Conexinas/fisiologia , Células HEK293 , Desacetilase 6 de Histona/genética , Humanos , Células Jurkat , Lisina/genética , Lisina/metabolismo , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Técnicas de Patch-Clamp , Receptores Adrenérgicos alfa 1/genética , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
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