RESUMO
Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.
Assuntos
Aldeído Desidrogenase/química , Proteínas de Bactérias/química , Doenças das Plantas/microbiologia , Pseudomonas syringae/enzimologia , Aldeído Desidrogenase/genética , Proteínas de Bactérias/genética , Cristalografia por Raios X , Pseudomonas syringae/genéticaRESUMO
BACKGROUND: Posttraumatic stress disorder (PTSD) is associated with increased risk for morbidity and mortality, which may be mediated through elevated inflammation. In contrast, social support appears to protect against morbidity and mortality, reduce levels of inflammation, and improve PTSD outcomes. METHODS: We examined relationships among social isolation, perceived social support, and inflammation in Veterans Affairs (VA) patients with and without PTSD. Our sample included 735 (35% PTSD+) participants from the Mind Your Heart Study (mean age = 58 ± 11; 94% male). Social isolation was assessed with the Berkman Syme Social Network Index; perceived social support with the Multidimensional Scale of Perceived Social Support; and PTSD with the Clinician Administered PTSD Scale. Inflammation was indexed by high sensitivity C-reactive protein, white blood cell count, and fibrinogen. Hierarchical linear regression was used to examine associations between social measures and inflammation. PROCESS was used to examine the interactive effects of social relationships and PTSD on inflammation. RESULTS: Social isolation, but not low perceived social support, trended towards an association with elevated inflammation in the full sample. However, considering groups with and without PTSD separately, social isolation was significantly associated with all inflammatory markers among individuals without PTSD, but not among those with PTSD. CONCLUSIONS: Social integration is associated with reduced inflammation in individuals without, but not with, PTSD. Socially integrated individuals with PTSD did not have lower levels of inflammatory markers than socially isolated individuals with PTSD.
Assuntos
Transtornos de Estresse Pós-Traumáticos , Veteranos , Idoso , Feminino , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Integração Social , Apoio SocialRESUMO
MoS2 nanoribbons with armchair-terminated edges are semiconductors suitable for the tuning of electronic and magnetic properties. Our first-principles density function calculations reveal that a variety of transition-metal atomic chains deposited on some of the ribbons is able to transform the semiconductors into half metals, allowing transport of 100% spin-polarized currents. Furthermore, we found that a Si atomic chain is equally capable of achieving half metallicity when adsorbed on the same nanoribbon. These results should be useful for spintronic application.
RESUMO
Depression and anxiety have been linked to elevated inflammation in cross-sectional and longitudinal studies. Yet, in terms of longitudinal studies, findings are inconsistent regarding whether depression predicts worsening inflammation or vice versa, and anxiety has been infrequently examined. Further, we know little about longitudinal relationships between inflammation and specific symptom profiles of depression and anxiety. The current study examined longitudinal associations between depression and anxiety symptoms and inflammation in 13,775 people (59% women, average ageâ¯=â¯67) participating in the Health and Retirement Study - a population-based study focused on older adults. High sensitivity C-reactive protein and depression and anxiety symptoms were measured at two time-points separated by four years. We used cross-lagged panel models to examine bidirectional relationships, and tested interactions with gender. We found that depressive symptoms predicted increasing inflammation for men, but not for women, and inflammation predicted worsening depression for women, but not for men. These gender differences were driven by somatic symptoms. Specifically, somatic symptoms predicted increasing inflammation for men only and were predicted by inflammation for women only. Regardless of gender, inflammation predicted worsening dysphoric symptoms of depression, and lack of positive affect predicted increasing inflammation over time. Anxiety was not associated with inflammation longitudinally. These findings indicate bidirectional relationships between depressive symptoms and inflammation, but not between anxiety symptoms and inflammation, and that the direction of these effects may differ by gender and type of depressive symptom.
Assuntos
Depressão/fisiopatologia , Inflamação/fisiopatologia , Fatores Sexuais , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiedade/fisiopatologia , Transtornos de Ansiedade/fisiopatologia , Proteína C-Reativa/análise , Estudos Transversais , Transtorno Depressivo/fisiopatologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-IdadeRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has single-digit 5-year survival rates at <7%. There is a dire need to improve pre-malignant detection methods and identify new therapeutic targets for abrogating PDAC progression. To this end, we mined our previously published pseudopodium-enriched (PDE) protein/phosphoprotein datasets to identify novel PDAC-specific biomarkers and/or therapeutic targets. We discovered that integrin alpha 1 (ITGA1) is frequently upregulated in pancreatic cancers and associated precursor lesions. Expression of ITGA1-specific collagens within the pancreatic cancer microenvironment significantly correlates with indicators of poor patient prognosis, and depleting ITGA1 from PDAC cells revealed that it is required for collagen-induced tumorigenic potential. Notably, collagen/ITGA1 signaling promotes the survival of ALDH1-positive stem-like cells and cooperates with TGFß to drive gemcitabine resistance. Finally, we report that ITGA1 is required for TGFß/collagen-induced EMT and metastasis. Our data suggest that ITGA1 is a new diagnostic biomarker and target that can be leveraged to improve patient outcomes.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Cadeias alfa de Integrinas/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Família Aldeído Desidrogenase 1 , Animais , Antimetabólitos Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Colágeno/genética , Colágeno/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Transição Epitelial-Mesenquimal , Humanos , Cadeias alfa de Integrinas/antagonistas & inibidores , Cadeias alfa de Integrinas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Transdução de Sinais , Análise Serial de Tecidos , Fator de Crescimento Transformador beta/farmacologia , Microambiente Tumoral/genética , GencitabinaRESUMO
Mounting evidence highlights specific forms of psychological stress as risk factors for ill health. Particularly strong evidence indicates that childhood adversity and adulthood trauma exposure increase risk for physical and psychiatric disorders, and there is emerging evidence that inflammation may play a key role in these relationships. In a population-based sample from the Health and Retirement Study (n=11,198, mean age 69 ± 10), we examine whether childhood adversity, adulthood trauma, and the interaction between them are associated with elevated levels of the systemic inflammatory marker high sensitivity C-reactive protein (hsCRP). All models were adjusted for age, gender, race, education, and year of data collection, as well as other possible confounds in follow-up sensitivity analyses. In our sample, 67% of individuals had experienced at least one traumatic event during adulthood, and those with childhood adversity were almost three times as likely to have experienced trauma as an adult. Childhood adversities and adulthood traumas were independently associated with elevated levels of hsCRP (ß=0.03, p=0.01 and ß=0.05, p<0.001, respectively). Those who had experienced both types of stress had higher levels of hsCRP than those with adulthood trauma alone, Estimate=-0.06, 95% CI [-0.003, -0.12], p=0.04, but not compared to those with childhood adversity alone, Estimate=-0.06, 95% CI [0.03, -0.16], p=0.19. There was no interaction between childhood and adulthood trauma exposure. To our knowledge, this is the first study to examine adulthood trauma exposure and inflammation in a large population-based sample, and the first to explore the interaction of childhood adversity and adulthood trauma with inflammation. Our study demonstrates the prevalence of trauma-related inflammation in the general population and suggests that childhood adversity and adulthood trauma are independently associated with elevated inflammation.
Assuntos
Proteína C-Reativa/metabolismo , Estresse Psicológico/sangue , Transtornos Relacionados a Trauma e Fatores de Estresse/sangue , Idoso , Biomarcadores/sangue , Estudos Transversais , Feminino , Humanos , Inflamação/sangue , Inflamação/epidemiologia , Estudos Longitudinais , Masculino , Transtornos Mentais/sangue , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Fatores Socioeconômicos , Estresse Psicológico/epidemiologia , Transtornos Relacionados a Trauma e Fatores de Estresse/epidemiologia , Estados Unidos/epidemiologiaRESUMO
While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their preactivation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome wide as cells recover from DNA damage, suggesting the general nature of this mechanism.
Assuntos
Dano ao DNA , Histona Acetiltransferases/metabolismo , Regiões Promotoras Genéticas , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Estudo de Associação Genômica Ampla , Histona Acetiltransferases/genética , Humanos , Fosforilação/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Peripheral artery disease (PAD) currently affects approximately 27 million patients in Europe and North America, and if untreated, may progress to the stage of critical limb ischemia (CLI), which has implications for amputation and potential mortality. Unfortunately, few therapies exist for treating the ischemic skeletal muscle in these conditions. Biomaterials have been used to increase cell transplant survival as well as deliver growth factors to treat limb ischemia; however, existing materials do not mimic the native skeletal muscle microenvironment they are intended to treat. Furthermore, no therapies involving biomaterials alone have been examined. The goal of this study was to develop a clinically relevant injectable hydrogel derived from decellularized skeletal muscle extracellular matrix and examine its potential for treating PAD as a stand-alone therapy by studying the material in a rat hindlimb ischemia model. We tested the mitogenic activity of the scaffold's degradation products using an in vitro assay and measured increased proliferation rates of smooth muscle cells and skeletal myoblasts compared to collagen. In a rat hindlimb ischemia model, the femoral artery was ligated and resected, followed by injection of 150 µL of skeletal muscle matrix or collagen 1 week post-injury. We demonstrate that the skeletal muscle matrix increased arteriole and capillary density, as well as recruited more desmin-positive and MyoD-positive cells compared to collagen. Our results indicate that this tissue-specific injectable hydrogel may be a potential therapy for treating ischemia related to PAD, as well as have potential beneficial effects on restoring muscle mass that is typically lost in CLI.
Assuntos
Matriz Extracelular , Músculo Esquelético/transplante , Neovascularização Fisiológica , Doença Arterial Periférica/terapia , Animais , Desmina/metabolismo , Modelos Animais de Doenças , Artéria Femoral/lesões , Membro Posterior/lesões , Humanos , Isquemia , Músculo Esquelético/citologia , Especificidade de Órgãos , RatosRESUMO
Profound chromatin changes occur during mitosis to allow for gene silencing and chromosome segregation followed by reactivation of memorized transcription states in daughter cells. Using genome-wide sequencing, we found H2A.Z-containing +1 nucleosomes of active genes shift upstream to occupy TSSs during mitosis, significantly reducing nucleosome-depleted regions. Single-molecule analysis confirmed nucleosome shifting and demonstrated that mitotic shifting is specific to active genes that are silenced during mitosis and, thus, is not seen on promoters, which are silenced by methylation or mitotically expressed genes. Using the GRP78 promoter as a model, we found H3K4 trimethylation is also maintained while other indicators of active chromatin are lost and expression is decreased. These key changes provide a potential mechanism for rapid silencing and reactivation of genes during the cell cycle.
Assuntos
Inativação Gênica , Histonas/metabolismo , Mitose/genética , Nucleossomos/metabolismo , Acetilação , Fator de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Metilação de DNA/fisiologia , DNA Polimerase II/metabolismo , Chaperona BiP do Retículo Endoplasmático , Fase G1/genética , Expressão Gênica/genética , Genes p16/fisiologia , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Metilação , Modelos Genéticos , Fosforilação/fisiologia , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fase de Repouso do Ciclo Celular/genética , Análise de Sequência de DNA , Proteína de Ligação a TATA-Box/metabolismo , Sítio de Iniciação de Transcrição/fisiologia , Quinase 1 Polo-LikeRESUMO
Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.
Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Nucleossomos/enzimologia , Linhagem Celular , Cromatina , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , DNA Metiltransferase 3BRESUMO
Epigenetic reprogramming is commonly observed in cancer, and is hypothesized to involve multiple mechanisms, including DNA methylation and Polycomb repressive complexes (PRCs). Here we devise a new experimental and analytical strategy using customized high-density tiling arrays to investigate coordinated patterns of gene expression, DNA methylation, and Polycomb marks which differentiate prostate cancer cells from their normal counterparts. Three major changes in the epigenomic landscape distinguish the two cell types. Developmentally significant genes containing CpG islands which are silenced by PRCs in the normal cells acquire DNA methylation silencing and lose their PRC marks (epigenetic switching). Because these genes are normally silent this switch does not cause de novo repression but might significantly reduce epigenetic plasticity. Two other groups of genes are silenced by either de novo DNA methylation without PRC occupancy (5mC reprogramming) or by de novo PRC occupancy without DNA methylation (PRC reprogramming). Our data suggest that the two silencing mechanisms act in parallel to reprogram the cancer epigenome and that DNA hypermethylation may replace Polycomb-based repression near key regulatory genes, possibly reducing their regulatory plasticity.
Assuntos
Metilação de DNA , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Reprogramação Celular , Ilhas de CpG/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Masculino , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Células-Tronco/metabolismoRESUMO
Epigenetic silencing of tumor suppressor genes is generally thought to involve DNA cytosine methylation, covalent modifications of histones, and chromatin compaction. Here, we show that silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells involves distinct changes in nucleosomal occupancy. Three nucleosomes, almost completely absent from the start sites in normal cells, are present on the methylated and silenced promoter, suggesting that epigenetic silencing may be accomplished by the stable placement of nucleosomes into previously vacant positions. Activation of the promoter by demethylation with 5-aza-2'-deoxycytidine involves nucleosome eviction. Epigenetic silencing of tumor suppressor genes may involve heritable changes in nucleosome occupancy enabled by cytosine methylation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Ilhas de CpG , Epigênese Genética , Inativação Gênica , Genes Supressores de Tumor , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Citosina/metabolismo , Metilação de DNA , Desoxirribonuclease I/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Genéticos , Proteína 1 Homóloga a MutL , Regiões Promotoras GenéticasRESUMO
Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (approximately 25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5' regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes.
Assuntos
Metilação de DNA , Genoma Humano , Histonas/metabolismo , Transcrição Gênica , Acetilação , Humanos , Reação em Cadeia da PolimeraseRESUMO
Epigenetic modifications of cytosine residues in DNA and the amino termini of histone proteins have emerged as key mechanisms in chromatin remodeling, impacting both the transcriptional regulation and the establishment of chromosomal domains. Using the chromatin immunoprecipitation (ChIP) assay, we demonstrate that aberrantly silenced genes in cancer cells exhibit a heterochromatic structure that is characterized by histone H3 lysine 9 (H3-K9) hypermethylation and histone H3 lysine 4 (H3-K4) hypomethylation. This aberrant heterochromatin is incompatible with transcriptional initiation but does not inhibit elongation by RNA polymerase II. H3-K9 methylation may, therefore, play a role in the silencing of tumor-suppressor genes in cancer. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), previously known for its ability to inhibit cytosine methylation, induced a rapid and substantial remodeling of the heterochromatic domains of the p14ARF/p16INK4a locus in T24 bladder cancer cells, reducing levels of dimethylated H3-K9 and increasing levels of dimethylated H3-K4 at this locus. In addition, 5-Aza-CdR increased acetylation and H3-K4 methylation at the unmethylated p14 promoter, suggesting it can induce chromatin remodeling independently of its effects on cytosine methylation.