RESUMO
SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.
Assuntos
Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sirtuínas/metabolismo , Animais , Respiração Celular , Transportador de Glucose Tipo 1 , Glicólise , Camundongos , Camundongos Knockout , Sirtuínas/genéticaRESUMO
The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Proteínas de Ciclo Celular/genética , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Chaperonas Moleculares/genética , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
BACKGROUND: Individuals with extreme food avoidance such as Avoidant Restrictive Food Intake Disorder (ARFID) experience impairing physical and mental health consequences from nutrition of insufficient variety or/and quantity. Identifying mechanisms contributing to food avoidance is essential to develop effective interventions. Anxiety figures prominently in theoretical models of food avoidance; however, there is limited evidence that repeated exposures to foods increases approach behavior in ARFID. Studying disgust, and relationships between disgust and anxiety, may offer novel insights, as disgust is functionally associated with avoidance of contamination from pathogens (as may occur via ingestion) and is largely resistant to extinction. METHOD: This exploratory, cross-sectional study included data from 1,644 adults who completed an online questionnaire. Participant responses were used to measure ARFID classification, picky eating, sensory sensitivity, disgust, and anxiety. Structural equation modeling tested a measurement model of latent disgust and anxiety factors as measured by self-reported frequency of disgust and anxiety reactions. Mediational models were used to explore causal ordering. RESULTS: A latent disgust factor was more strongly related to severity of picky eating (B ≈ 0.4) and ARFID classification (B ≈ 0.6) than the latent anxiety factor (B ≈ 0.1). Disgust partially mediated the association between anxiety and picky eating and fully mediated the association between anxiety and ARFID. Models testing the reverse causal ordering demonstrated poorer fit. Findings suggest anxiety may be associated with food avoidance in part due to increased disgust. CONCLUSIONS: Disgust may play a prominent role in food avoidance. Findings may inform novel approaches to treatment.
Assuntos
Asco , Ingestão de Alimentos/psicologia , Transtornos da Alimentação e da Ingestão de Alimentos/psicologia , Adolescente , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Cellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. Butyryl-CoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.
Assuntos
Acil Coenzima A/metabolismo , Tecido Adiposo/metabolismo , Dieta Hiperlipídica , Histonas/metabolismo , Fígado/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetilação , Acil Coenzima A/análise , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Deleção de Genes , Histonas/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/metabolismoRESUMO
Multiple substrate enzymes present a particular challenge when it comes to understanding their activity in a complex system. Although a single target may be easy to model, it does not always present an accurate representation of what that enzyme will do in the presence of multiple substrates simultaneously. Therefore, there is a need to find better ways to both study these enzymes in complicated systems, as well as accurately describe the interactions through kinetic parameters. This review looks at different methods for studying multiple substrate enzymes, as well as explores options on how to most accurately describe an enzyme's activity within these multi-substrate systems. Identifying and defining this enzymatic activity should help clear the way to using in vitro systems to accurately predicting the behavior of multi-substrate enzymes in vivo. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.
Assuntos
Ensaios Enzimáticos/métodos , Enzimas/metabolismo , Algoritmos , Biocatálise , Enzimas/química , Cinética , Modelos Químicos , Especificidade por SubstratoRESUMO
OBJECTIVE: The diagnostic criterion disturbance in the experience of the body remains a poorly understood and persistent feature of anorexia nervosa (AN). Increased sophistication in understanding the structure of the insular cortex-a neural structure that receives and integrates visceral sensations with action and meaning-may elucidate the nature of this disturbance. We explored age, weight status, illness severity, and self-reported body dissatisfaction associations with insular cortex volume. METHODS: Structural magnetic resonance imaging data were collected from 21 adolescents with a history of AN and 20 age-, sex-, and body mass index-matched controls. Insular cortical volumes (bilateral anterior and posterior regions) were identified using manual tracing. RESULTS: Volumes of the right posterior insula demonstrated the following: (a) a significant age by clinical status interaction (ß = -0.018 [0.008]; t = 2.32, p = .02) and (b) larger volumes were associated with longer duration of illness (r = 0.48, p < .04). In contrast, smaller volumes of the right anterior insula were associated with longer duration of illness (r = -0.50, p < .03). The associations of insular volume with body dissatisfaction were of moderate effect size and also of opposite direction, but a statistical trend in right posterior (r = 0.40, p < .10 in right posterior; r = -0.49, p < .04 in right anterior). CONCLUSIONS: In this exploratory study, findings of atypical structure of the right posterior insular cortex point to the importance of future work investigating the role of visceral afferent signaling in understanding disturbance in body experience in AN.
Assuntos
Anorexia Nervosa/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Adolescente , Fatores Etários , Criança , Feminino , Humanos , Imageamento por Ressonância Magnética , Fatores de TempoRESUMO
Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation.
Assuntos
Reparo do DNA , Proteínas de Drosophila/metabolismo , Raios gama , Histonas/metabolismo , Transcrição Gênica/efeitos da radiação , Acetilação , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , MutaçãoRESUMO
The histone acetyltransferase (HAT) enzymes p300 and CBP are closely related paralogs that serve as transcriptional coactivators and have been found to be dysregulated in cancer and other diseases. p300/CBP is a multidomain protein and possesses a highly conserved bromodomain that has been shown to bind acetylated Lys residues in both proteins and various small molecules, including I-CBP112 and CBP30. Here we show that the ligand I-CBP112 can stimulate nucleosome acetylation up to 3-fold while CBP30 does not. Activation of p300/CBP by I-CBP112 is not observed with the isolated histone H3 substrate but requires a nucleosome substrate. I-CBP112 does not impact nucleosome acetylation by the isolated p300 HAT domain, and the effects of I-CBP112 on p300/CBP can be neutralized by CBP30, suggesting that I-CBP112 likely allosterically activates p300/CBP through bromodomain interactions. Using mass spectrometry and Western blots, we have found that I-CBP112 particularly stimulates acetylation of Lys18 of histone H3 (H3K18) in nucleosomes, an established in vivo site of p300/CBP. In addition, we show that I-CBP112 enhances H3K18 acetylation in acute leukemia and prostate cancer cells in a concentration range commensurate with its antiproliferative effects. Our findings extend the known pharmacology of bromodomain ligands in the regulation of p300/CBP and suggest a novel approach to modulating histone acetylation in cancer.
Assuntos
Compostos de Bromo/farmacologia , Proteína p300 Associada a E1A/metabolismo , Leucemia/patologia , Nucleossomos/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Histonas/metabolismo , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Ligação Proteica , Conformação Proteica , Células Tumorais CultivadasRESUMO
How protein-protein interactions regulate and alter histone modifications is a major unanswered question in epigenetics. The histone acetyltransferase p300 binds thymine DNA glycosylase (TDG); utilizing mass spectrometry to measure site-specific changes in histone acetylation, we found that the absence of TDG in mouse embryonic fibroblasts leads to a reduction in the rate of histone acetylation. We demonstrate that TDG interacts with the CH3 domain of p300 to allosterically promote p300 activity to specific lysines on histone H3 (K18 and K23). However, when TDG concentrations approach those of histones, TDG acts as a competitive inhibitor of p300 histone acetylation. These results suggest a mechanism for how histone acetylation is fine-tuned via interaction with other proteins, while also highlighting a connection between regulators of two important biological processes: histone acetylation and DNA repair/demethylation.
Assuntos
Reparo do DNA , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Timina DNA Glicosilase/metabolismo , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Camundongos , Camundongos Knockout , Timina DNA Glicosilase/genéticaRESUMO
The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.
Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 8/metabolismo , Proliferação de Células , Humanos , Mitocôndrias/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas/metabolismoRESUMO
We have a limited understanding of the site specificity of multi-subunit lysine acetyltransferase (KAT) complexes for histone-based substrates, especially in regards to the different complexes formed during nucleosome assembly. Histone complexes could be a major factor in determining the acetylation specificity of KATs. In the present study, we utilized a label-free quantitative MS-based method to determine the site specificity of acetylation catalysed by Piccolo NuA4 on (H3/H4)2 tetramer, tetramer bound DNA (tetrasome) and nucleosome core particle (NCP). Our results show that Piccolo NuA4 can acetylate multiple lysine residues on these three histone complexes, of which NCP is the most favourable, (H3/H4)2 tetramer is the second and tetrasome is the least favourable substrate for Piccolo NuA4 acetylation. Although Piccolo NuA4 preferentially acetylates histone H4 (H4K12), the site specificity of the enzyme is altered with different histone complex substrates. Our results show that before nucleosome assembly is complete, H3K14 specificity is almost equal to that of H4K12 and DNA-histone interactions suppress the acetylation ability of Piccolo NuA4. These data suggest that the H2A/H2B dimer could play a critical role in the increase in acetylation specificity of Piccolo NuA4 for NCP. This demonstrates that histone complex formation can alter the acetylation preference of Piccolo NuA4. Such findings provide valuable insight into regulating Piccolo NuA4 specificity by modulating chromatin dynamics and in turn manipulating gene expression.
Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Xenopus/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Acetiltransferases , Animais , Montagem e Desmontagem da Cromatina , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histonas/química , Histonas/genética , Cinética , Lisina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nucleossomos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMO
CBX5, CBX1, and CBX3 (HP1α, ß, and γ, respectively) play an evolutionarily conserved role in the formation and maintenance of heterochromatin. In addition, CBX5, CBX1, and CBX3 may also participate in transcriptional regulation of genes. Recently, CBX3 binding to the bodies of a subset of genes has been observed in human and murine cells. However, the generality of this phenomenon and the role CBX3 may play in this context are unknown. Genome-wide localization analysis reveals CBX3 binding at genic regions, which strongly correlates with gene activity across multiple cell types. Depletion of CBX3 resulted in down-regulation of a subset of target genes. Loss of CBX3 binding leads to a more dramatic accumulation of unspliced nascent transcripts. In addition, we observed defective recruitment of splicing factors, including SNRNP70, to CBX3 target genes. Collectively, our data suggest a role for CBX3 in aiding in efficient cotranscriptional RNA processing.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Genoma Humano , Heterocromatina/metabolismo , Processamento Pós-Transcricional do RNA , Sítios de Ligação , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Éxons , Regulação da Expressão Gênica , Células HCT116 , Heterocromatina/genética , Humanos , Células K562 , Ligação Proteica , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.
Assuntos
Histonas/metabolismo , Espectrometria de Massas/métodos , Acetilação , Cromatografia Líquida de Alta Pressão , Histonas/química , Cinética , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Recent genome-wide studies have shown that approximately 30% of diffuse large B-cell lymphoma (DLBCL) cases harbor mutations in the histone acetyltransferase (HAT) coactivators p300 or CBP. The majority of these mutations reduce or eliminate the catalytic HAT activity. We previously demonstrated that the human DLBCL cell line RC-K8 expresses a C-terminally truncated, HAT-defective p300 protein (p300ΔC-1087), whose expression is essential for cell proliferation. METHODS: Using results from large-scale DLBCL studies, we have identified and characterized a second C-terminally truncated, HAT-defective p300 mutant, p300ΔC-820, expressed in the SUDHL2 DLBCL cell line. Properties of p300ΔC-820 were characterized in the SUDHL2 DLBCL cell line by Western blotting, co-immunoprecipitation, and shRNA gene knockdown, as well by using cDNA expression vectors for p300ΔC-820 in pull-down assays, transcriptional reporter assays, and immunofluorescence experiments. A mass spectrometry-based method was used to compare the histone acetylation profile of DLBCL cell lines expressing various levels of wild-type p300. RESULTS: We show that the SUDHL2 cell line expresses a C-terminally truncated, HAT-defective form of p300 (p300ΔC-820), but no wild-type p300. The p300ΔC-820 protein has a wild-type ability to localize to subnuclear "speckles," but has a reduced ability to enhance transactivation by transcription factor REL. Knockdown of p300ΔC-820 in SUDHL2 cells reduced their proliferation and soft agar colony-forming ability. In RC-K8 cells, knockdown of p300ΔC-1087 resulted in increased expression of mRNA and protein for REL target genes A20 and IκBα, two genes that have been shown to limit the growth of RC-K8 cells when overexpressed. Among a panel of B-lymphoma cell lines, low-level expression of full-length p300 protein, which is characteristic of the SUDHL2 and RC-K8 cells, was associated with decreased acetylation of histone H3 at lysines 14 and 18. CONCLUSIONS: The high prevalence of p300 mutations in DLBCL suggests that HAT-deficient p300 activity defines a subtype of DLBCL, which we have investigated using human DLBCL cell lines RC-K8 and SUDHL2. Our results suggest that truncated p300 proteins contribute to DLBCL cell growth by affecting the expression of specific genes, perhaps through a mechanism that involves alterations in global histone acetylation.
Assuntos
Proliferação de Células , Regulação Neoplásica da Expressão Gênica/genética , Linfoma Difuso de Grandes Células B/genética , Fatores de Transcrição de p300-CBP/genética , Acetilação , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Histonas/genética , Histonas/metabolismo , Humanos , Imunoprecipitação , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TranscriptomaRESUMO
The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small-molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a genome-wide short hairpin RNA screen for genes that are lethal in combination with p53 activation by Nutlin-3, which showed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors also enable Nutlin-3 to kill tumor spheroids. These results identify new pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes p53 , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Genes Letais , Genes Sintéticos , Humanos , Imidazóis/metabolismo , Piperazinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Background: Ecological momentary assessment (EMA) as a real-time data collection method can provide insight into the daily experiences of family caregivers. Purpose: This systematic review aimed to synthesize studies involving EMA completed by family caregivers of adults with chronic conditions. Methods: A systematic search was conducted within six databases for articles published from the inception of the database through September 2023. We extracted the characteristics of the included studies and data on EMA-specific methods to determine the quality of the included studies. Results: A total of 12 studies involving EMA completed by family caregivers of adults with chronic conditions were identified, with almost all studies focused on caregivers of persons with Alzheimer's or dementia-related conditions. The average compliance rate across the included studies was 75%, below the recommended rate. In addition, most of the included studies did not collect the family caregivers' daily activities and care contexts in their responses (i.e., affect, stress, well-being, care demand, and fatigue) to the EMA prompts. Discussion: This review showed that using EMA to collect information on family caregivers of adults with chronic health conditions appeared feasible and acceptable. However, the methodology or design of using EMA to collect caregiver information in this population is still preliminary. The limited number of existing studies that have used EMA to capture the daily experiences of family caregivers does not provide key information that could improve understanding of caregivers' emotional experiences and well-being in real-life situations. We identified gaps in the literature that warrant additional EMA studies for this population.
RESUMO
Although p300 and CBP lysine acetyltransferases are often treated interchangeably, the inability of one enzyme to compensate for the loss of the other suggests unique roles for each. As these deficiencies coincide with aberrant levels of histone acetylation, we hypothesized that the key difference between p300 and CBP activity is differences in their specificity/selectivity for lysines within the histones. Utilizing a label-free, quantitative mass spectrometry based technique, we determined the kinetic parameters of both CBP and p300 at each lysine of H3 and H4, under conditions we would expect to encounter in the cell (either limiting acetyl-CoA or histone). Our results show that while p300 and CBP acetylate many common residues on H3 and H4, they do in fact possess very different specificities, and these specificities are dependent on whether histone or acetyl-CoA is limiting. Steady-state experiments with limiting H3 demonstrate that both CBP and p300 acetylate H3K14, H3K18, H3K23, with p300 having specificities up to 10¹°-fold higher than CBP. Utilizing tetramer as a substrate, both enzymes also acetylate H4K5, H4K8, H4K12, and H4K16. With limiting tetramer, CBP displays higher specificities, especially at H3K18, where CBP specificity is 10³²-fold higher than p300. With limiting acetyl-CoA, p300 has the highest specificity at H4K16, where specificity is 10¹8-fold higher than CBP. This discovery of unique specificity for targets of CBP- vs p300-mediated acetylation of histone lysine residues presents a new model for understanding their respective biological roles and possibly an opportunity for selective therapeutic intervention.
Assuntos
Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Humanos , Especificidade por Substrato , Fatores de Transcrição de p300-CBP/genéticaRESUMO
The p53 tumor suppressor is embedded in a large gene network controlling diverse cellular and organismal phenotypes. Multiple signaling pathways converge onto p53 activation, mostly by relieving the inhibitory effects of its repressors, MDM2 and MDM4. In turn, signals originating from increased p53 activity diverge into distinct effector pathways to deliver a specific cellular response to the activating stimuli. Much attention has been devoted to dissecting how the various input pathways trigger p53 activation and how the activity of the p53 protein itself can be modulated by a plethora of co-factors and post-translational modifications. In this review we will focus instead on the multiple configurations of the effector pathways. We will discuss how p53-generated signals are transmitted, amplified, resisted and eventually integrated by downstream gene circuits operating at the transcriptional, post-transcriptional and post-translational levels. We will also discuss how context-dependent variations in these gene circuits define the cellular response to p53 activation and how they may impact the clinical efficacy of p53-based targeted therapies.
Assuntos
Genes p53 , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Humanos , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
The eukaryotic green algae, Chlamydomonas reinhardtii has been shown to be capable of producing a variety of recombinant proteins, but the true potential of this platform remains largely unexplored. To assess the potential of algae for the production of novel recombinant proteins, we generated a series of chimeric proteins containing a single chain antibody (scFv) targeting the B-cell surface antigen CD22, genetically fused to the eukaryotic ribosome inactivating protein, gelonin, from Gelonium multiflorm. These unique molecules, termed immunotoxins, are encoded as a single gene that produces an antibody--toxin chimeric protein capable of delivering a cytotoxic molecule to targeted B-cells. We show that the addition of an Fc domain of a human IgG1 to these fusion proteins results in the production of assembled dimeric immunotoxins, containing two cell binding scFvs and two gelonin molecules. Additionally, we demonstrate that these algal expressed proteins are capable of binding and reducing the viability of B-cell lymphomas, while treatment of T-cells, that lack the CD22 antigen, had no impact on cell viability. Since other protein expression platforms are incapable of folding and accumulating these complex immunotoxins as soluble and enzymatically active proteins, our studies document a novel and efficient method for immunotoxin production.
Assuntos
Antineoplásicos/metabolismo , Chlamydomonas reinhardtii/metabolismo , Imunotoxinas/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlamydomonas reinhardtii/genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunotoxinas/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Análise de Sequência de DNA , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
OBJECTIVES: We examined the empirical link between money mismanagement and subsequent homelessness among veterans. METHODS: We used a random sample of Iraq and Afghanistan War era veterans from the National Post-Deployment Adjustment Survey in 2009-2011. RESULTS: Veterans were randomly selected from a roster of all US military service members in Operation Iraqi Freedom or Operation Enduring Freedom who were separated from active duty or in the Reserves/National Guard. Veterans (n = 1090) from 50 states and all military branches completed 2 waves of data collection 1 year apart (79% retention rate). Thirty percent reported money mismanagement (e.g., bouncing or forging a check, going over one's credit limit, falling victim to a money scam in the past year). Multivariate analysis revealed money mismanagement (odds ratio [OR] = 4.09, 95% CI = 1.87, 8.94) was associated with homelessness in the next year, as were arrest history (OR = 2.65, 95% CI = 1.33, 5.29), mental health diagnosis (OR = 2.59, 95% CI = 1.26, 5.33), and income (OR = 0.30, 95% CI = 0.13, 0.71). CONCLUSIONS: Money mismanagement, reported by a substantial number of veterans, was related to a higher rate of subsequent homelessness. The findings have implications for policymakers and clinicians, suggesting that financial education programs offered by the US Departments of Defense and Veterans Affairs may be targeted to effectively address veteran homelessness.