RESUMO
A multitude of histone chaperones are required to support histones from their biosynthesis until DNA deposition. They cooperate through the formation of histone co-chaperone complexes, but the crosstalk between nucleosome assembly pathways remains enigmatic. Using exploratory interactomics, we define the interplay between human histone H3-H4 chaperones in the histone chaperone network. We identify previously uncharacterized histone-dependent complexes and predict the structure of the ASF1 and SPT2 co-chaperone complex, expanding the role of ASF1 in histone dynamics. We show that DAXX provides a unique functionality to the histone chaperone network, recruiting histone methyltransferases to promote H3K9me3 catalysis on new histone H3.3-H4 prior to deposition onto DNA. Hereby, DAXX provides a molecular mechanism for de novo H3K9me3 deposition and heterochromatin assembly. Collectively, our findings provide a framework for understanding how cells orchestrate histone supply and employ targeted deposition of modified histones to underpin specialized chromatin states.
Assuntos
Chaperonas de Histonas , Histonas , Humanos , Histonas/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Nucleossomos/genética , Proteínas de Ciclo Celular/metabolismo , DNA , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismoRESUMO
Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Camptotecina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Células HeLa , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Inibidores da Topoisomerase I/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
Toll-like receptor (TLR) activation induces inflammatory responses in macrophages by activating temporally defined transcriptional cascades. Whether concurrent changes in the cellular metabolism that occur upon TLR activation influence the quality of the transcriptional responses remains unknown. Here, we investigated how macrophages adopt their metabolism early after activation to regulate TLR-inducible gene induction. Shortly after TLR4 activation, macrophages increased glycolysis and tricarboxylic acid (TCA) cycle volume. Metabolic tracing studies revealed that TLR signaling redirected metabolic fluxes to generate acetyl-Coenzyme A (CoA) from glucose resulting in augmented histone acetylation. Signaling through the adaptor proteins MyD88 and TRIF resulted in activation of ATP-citrate lyase, which in turn facilitated the induction of distinct LPS-inducible gene sets. We postulate that metabolic licensing of histone acetylation provides another layer of control that serves to fine-tune transcriptional responses downstream of TLR activation. Our work highlights the potential of targeting the metabolic-epigenetic axis in inflammatory settings.
Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Acetilcoenzima A/metabolismo , Histonas/metabolismo , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Acetilação , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Ciclo do Ácido Cítrico/fisiologia , Glicólise/fisiologia , Humanos , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Transcrição Gênica/genéticaRESUMO
The mitochondrial oxidative phosphorylation system comprises complexes assembled from subunits derived from mitochondrial and nuclear gene expression. Both genetic systems are coordinated by feedback loops, which control the synthesis of specific mitochondrial encoded subunits. Here, we studied how this occurs in the case of cytochrome b, a key subunit of mitochondrial complex III. Our data suggest the presence of a molecular rheostat consisting of two translational activators, Cbp3-Cbp6 and Cbs1, which operates at the mitoribosomal tunnel exit to connect translational output with assembly efficiency. When Cbp3-Cbp6 is engaged in assembly of cytochrome b, Cbs1 binds to the tunnel exit to sequester the cytochrome b-encoding mRNA, repressing its translation. After mediating complex III assembly, binding of Cbp3-Cbp6 to the tunnel exit replaces Cbs1 and the bound mRNA to permit cytochrome b synthesis. Collectively, the data indicate the molecular wiring of a feedback loop to regulate synthesis of a mitochondrial encoded protein.
Assuntos
Regulação da Expressão Gênica , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Citocromos b/biossíntese , Citocromos b/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Chaperonas Moleculares/metabolismo , RNA Mensageiro/análise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/metabolismoRESUMO
Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.
Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Fosforilação Oxidativa , Biossíntese de Proteínas/genética , Saccharomyces cerevisiae/genéticaRESUMO
Chromatin-modifying enzymes have long been proposed to be the authors of an epigenetic language, but the origin and meaning of the messages they write in chromatin are still mysterious. Recent studies suggesting that the effects of diet can be passed on epigenetically to offspring add weight to the idea that histones act as metabolic sensors, converting changes in metabolism into stable patterns of gene expression. The challenge will now be to understand how localized fluctuations in levels of metabolites control chromatin modifiers in space and time, translating a dynamic metabolic state into a histone map.
Assuntos
Epigênese Genética , Eucariotos/genética , Eucariotos/metabolismo , Redes e Vias Metabólicas , Montagem e Desmontagem da Cromatina , Código das Histonas , Histonas/metabolismo , NAD/metabolismoRESUMO
The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.
Assuntos
RNA Polimerase II/genética , RNA Mensageiro/biossíntese , Terminação da Transcrição Genética/fisiologia , Transcrição Gênica/fisiologia , Tirosina/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Humanos , Mutação/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/genéticaRESUMO
Highly specialized cells are fundamental for the proper functioning of complex organs. Variations in cell-type-specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high costs and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline to the mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of the dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method's general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.
RESUMO
Acetylation of lysine 16 of histone H4 (H4K16ac) stands out among the histone modifications, because it decompacts the chromatin fiber. The metazoan acetyltransferase MOF (KAT8) regulates transcription through H4K16 acetylation. Antibody-based studies had yielded inconclusive results about the selectivity of MOF to acetylate the H4 N-terminus. We used targeted mass spectrometry to examine the activity of MOF in the male-specific lethal core (4-MSL) complex on nucleosome array substrates. This complex is part of the Dosage Compensation Complex (DCC) that activates X-chromosomal genes in male Drosophila. During short reaction times, MOF acetylated H4K16 efficiently and with excellent selectivity. Upon longer incubation, the enzyme progressively acetylated lysines 12, 8 and 5, leading to a mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains substrate-bound and continues to acetylate more N-terminal H4 lysines in a processive manner. The 4-MSL complex lacks non-coding roX RNA, a critical component of the DCC. Remarkably, addition of RNA to the reaction non-specifically suppressed H4 oligo-acetylation in favor of specific H4K16 acetylation. Because RNA destabilizes the MSL-nucleosome interaction in vitro we speculate that RNA accelerates enzyme-substrate turn-over in vivo, thus limiting the processivity of MOF, thereby increasing specific H4K16 acetylation.
Assuntos
Proteínas de Drosophila , Histona Acetiltransferases , Código das Histonas , Animais , Masculino , Acetilação , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas Nucleares , Nucleossomos/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.
Assuntos
Neoplasias Encefálicas , Glioma , Histonas , Criança , Humanos , Neoplasias Encefálicas/patologia , Reparo do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Glioma/patologia , Histonas/genética , Histonas/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
Cellular plasticity is crucial for adapting to ever-changing stimuli. As a result, cells consistently reshape their translatome, and, consequently, their proteome. The control of translational activity has been thoroughly examined at the stage of translation initiation. However, the regulation of ribosome speed in cells is widely unknown. In this study, we utilized a timed ribosome runoff approach, along with proteomics and transmission electron microscopy, to investigate global translation kinetics in cells. We found that ribosome speeds vary among various cell types, such as astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Of all cell types studied, mature cortical neurons exhibit the highest rate of translation. This finding is particularly remarkable because mature cortical neurons express the eukaryotic elongation factor 2 (eEF2) at lower levels than other cell types. Neurons solve this conundrum by inactivating a fraction of their ribosomes. As a result, the increase in eEF2 levels leads to a reduction of inactive ribosomes and an enhancement of active ones. Processes that alter the demand for active ribosomes, like neuronal excitation, cause increased inactivation of redundant ribosomes in an eEF2-dependent manner. Our data suggest a novel regulatory mechanism in which neurons dynamically inactivate ribosomes to facilitate translational remodeling. These findings have important implications for developmental brain disorders characterized by, among other things, aberrant translation.
Assuntos
Biossíntese de Proteínas , Ribossomos , Animais , Humanos , Ratos , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Proteins involved in cellular metabolism and molecular regulation can extend lifespan of various organisms in the laboratory. However, any improvement in aging would only provide an evolutionary benefit if the organisms were able to survive under non-ideal conditions. We have previously shown that Drosophila melanogaster carrying a loss-of-function allele of the acetyltransferase chameau (chm) has an increased healthy lifespan when fed ad libitum. Here, we show that loss of chm and reduction in its activity results in a substantial reduction in weight and a decrease in starvation resistance. This phenotype is caused by failure to properly regulate the genes and proteins required for energy storage and expenditure. The previously observed increase in survival time thus comes with the inability to prepare for and cope with nutrient stress. As the ability to survive in environments with restricted food availability is likely a stronger evolutionary driver than the ability to live a long life, chm is still present in the organism's genome despite its apparent negative effect on lifespan.
RESUMO
The largest subunit of RNA polymerase (Pol) II harbors an evolutionarily conserved C-terminal domain (CTD), composed of heptapeptide repeats, central to the transcriptional process. Here, we analyze the transcriptional phenotypes of a CTD-Δ5 mutant that carries a large CTD truncation in human cells. Our data show that this mutant can transcribe genes in living cells but displays a pervasive phenotype with impaired termination, similar to but more severe than previously characterized mutations of CTD tyrosine residues. The CTD-Δ5 mutant does not interact with the Mediator and Integrator complexes involved in the activation of transcription and processing of RNAs. Examination of long-distance interactions and CTCF-binding patterns in CTD-Δ5 mutant cells reveals no changes in TAD domains or borders. Our data demonstrate that the CTD is largely dispensable for the act of transcription in living cells. We propose a model in which CTD-depleted Pol II has a lower entry rate onto DNA but becomes pervasive once engaged in transcription, resulting in a defect in termination.
Assuntos
RNA Polimerase II , Transcrição Gênica , Humanos , RNA Polimerase II/metabolismo , Núcleo Celular/metabolismo , Mutação , FosforilaçãoRESUMO
The use of synthetic chemicals to selectively interfere with chromatin and the chromatin-bound proteome represents a great opportunity for pharmacological intervention. Recently, synthetic foldamers that mimic the charge surface of double-stranded DNA have been shown to interfere with selected protein-DNA interactions. However, to better understand their pharmacological potential and to improve their specificity and selectivity, the effect of these molecules on complex chromatin needs to be investigated. We therefore systematically studied the influence of the DNA mimic foldamers on the chromatin-bound proteome using an in vitro chromatin assembly extract. Our studies show that the foldamer efficiently interferes with the chromatin-association of the origin recognition complex in vitro and in vivo, which leads to a disturbance of cell cycle in cells treated with foldamers. This effect is mediated by a strong direct interaction between the foldamers and the origin recognition complex and results in a failure of the complex to organise chromatin around replication origins. Foldamers that mimic double-stranded nucleic acids thus emerge as a powerful tool with designable features to alter chromatin assembly and selectively interfere with biological mechanisms.
Assuntos
Biomimética , Montagem e Desmontagem da Cromatina , Ciclo Celular , Cromatina , DNA , Replicação do DNA , Complexo de Reconhecimento de Origem/metabolismo , Proteoma , Animais , Drosophila , Embrião não Mamífero/química , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismoRESUMO
The foundation for integrating mass spectrometry (MS)-based proteomics into systems medicine is the development of standardized start-to-finish and fit-for-purpose workflows for clinical specimens. An essential step in this pursuit is to highlight the common ground in a diverse landscape of different sample preparation techniques and liquid chromatography-mass spectrometry (LC-MS) setups. With the aim to benchmark and improve the current best practices among the proteomics MS laboratories of the CLINSPECT-M consortium, we performed two consecutive round-robin studies with full freedom to operate in terms of sample preparation and MS measurements. The six study partners were provided with two clinically relevant sample matrices: plasma and cerebrospinal fluid (CSF). In the first round, each laboratory applied their current best practice protocol for the respective matrix. Based on the achieved results and following a transparent exchange of all lab-specific protocols within the consortium, each laboratory could advance their methods before measuring the same samples in the second acquisition round. Both time points are compared with respect to identifications (IDs), data completeness, and precision, as well as reproducibility. As a result, the individual performances of participating study centers were improved in the second measurement, emphasizing the effect and importance of the expert-driven exchange of best practices for direct practical improvements.
Assuntos
Plasma , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Fluxo de Trabalho , Reprodutibilidade dos Testes , Plasma/químicaRESUMO
TMF1-regulated nuclear protein 1 (Trnp1) has been shown to exert potent roles in neural development affecting neural stem cell self-renewal and brain folding, but its molecular function in the nucleus is still unknown. Here, we show that Trnp1 is a low complexity protein with the capacity to phase separate. Trnp1 interacts with factors located in several nuclear membrane-less organelles, the nucleolus, nuclear speckles, and condensed chromatin. Importantly, Trnp1 co-regulates the architecture and function of these nuclear compartments in vitro and in the developing brain in vivo. Deletion of a highly conserved region in the N-terminal intrinsic disordered region abolishes the capacity of Trnp1 to regulate nucleoli and heterochromatin size, proliferation, and M-phase length; decreases the capacity to phase separate; and abrogates most of Trnp1 protein interactions. Thus, we identified Trnp1 as a novel regulator of several nuclear membrane-less compartments, a function important to maintain cells in a self-renewing proliferative state.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Neurais/metabolismo , Membrana Nuclear/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Camundongos , Membrana Nuclear/genética , Domínios ProteicosRESUMO
Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harness developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here, we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles (HMPs), in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted toward normality on morphological, molecular, and epigenetic levels. These results suggest that the cell cycle block by HUA alters stage-specific HMPs. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.
Assuntos
Epigênese Genética , Código das Histonas , Xenopus laevis/embriologia , Animais , Afidicolina/farmacologia , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/embriologia , Inibidores Enzimáticos/farmacologia , Hidroxiureia/farmacologiaRESUMO
BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.
Assuntos
Aterosclerose , Água Potável , Placa Aterosclerótica , Aminoácidos , Animais , Apolipoproteínas E , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Feminino , Homoarginina/farmacologia , Camundongos , Cadeias Pesadas de Miosina , Linfócitos T/metabolismoRESUMO
The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
Assuntos
RNA Polimerase II/química , RNA Polimerase II/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/metabolismo , Humanos , Mamíferos , Espectrometria de Massas , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fosforilação , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Aminoácidos , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI-IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI-IMS. This is achieved by combining information from IMS and shotgun proteomics (LC-MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC-MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.