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Discovery of interventions that delay or minimize age-related diseases is arguably the major goal of aging research. Conversely discovery of interventions based on phenotypic screens have often led to further elucidation of pathophysiological mechanisms. Although most hypotheses to explain lifespan focus on cell-autonomous processes, increasing evidence suggests that in multicellular organisms, neurons, particularly nutrient-sensing neurons, play a determinative role in lifespan and age-related diseases. For example, protective effects of dietary restriction and inactivation of insulin-like signaling increase lifespan and delay age-related diseases dependent on Creb-binding protein in GABA neurons, and Nrf2/Skn1 in just 2 nutrient-sensing neurons in C. elegans. Screens for drugs that increase lifespan also indicate that such drugs are predominantly active through neuronal signaling. Our own screens also indicate that neuroactive drugs also delay pathology in an animal model of Alzheimer's Disease, as well as inhibit cytokine production implicated in driving many age-related diseases. The most likely mechanism by which nutrient-sensing neurons influence lifespan and the onset of age-related diseases is by regulating metabolic architecture, particularly the relative rate of glycolysis vs. alternative metabolic pathways such as ketone and lipid metabolism. These results suggest that neuroactive compounds are a most promising class of drugs to delay or minimize age-related diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sepsis-induced inflammatory lung injury is a key factor causing failure of the lungs and other organs, as well as death, during sepsis. In the present study, a caecal ligation and puncture (CLP)-induced sepsis model was established to investigate the effect of β-catenin on sepsis-induced inflammatory lung injury and the corresponding underlying mechanisms. C57BL/6 mice were randomly divided into five groups, namely, the sham, CLP, β-catenin knockout (KO) + CLP, XAV-939 + CLP, and ICG-001 + CLP groups; the XAV-939 + CLP and ICG-001 + CLP groups were separately subjected to intraperitoneal injections of the β-catenin inhibitors XAV-939 and ICG-001 for 1 week preoperatively and 2 days postoperatively, respectively. Forty-eight hours after CLP, we measured β-catenin expression in lung tissues and evaluated mouse mortality, histopathological characteristics of hematoxylin and eosin (H&E)-stained lung tissues, serum cytokine (tumor necrosis factor [TNF]-α, interleukin [IL]-10, and IL-1β) levels, lung myeloperoxidase (MPO) activity, and the number of apoptotic cells in the lung tissues. Our results indicated that both the inhibition of β-catenin expression and blockage of β-catenin/CREB-binding protein (CBP) interactions by ICG-001 effectively decreased mouse mortality, alleviated pathological lung injury, and reduced the serum TNF-α, IL-10, and IL-1β levels, in addition to reducing the lung MPO activity and the number of apoptotic cells in lung tissues of the sepsis model mice. Therefore, it can be deduced that the β-catenin/CBP signaling axis participates in regulating sepsis-induced inflammatory lung injury.

Disruption of fetal growth results in severe consequences to human health, including increased fetal and neonatal morbidity and mortality, as well as potential lifelong health problems. Molecular mechanisms promoting fetal growth represent potential therapeutic strategies to treat and/or prevent fetal growth restriction (FGR). Here, we identify a previously unknown role for the mitogen-activated protein kinase kinase kinase 4 (MAP3K4) in promoting fetal and placental growth. We demonstrate that inactivation of MAP3K4 kinase activity causes FGR due in part to placental insufficiency. Significantly, MAP3K4 kinase-inactive mice display highly penetrant lethality prior to weaning and persistent growth reduction of surviving adults. Additionally, we elucidate molecular mechanisms by which MAP3K4 promotes growth through control of the insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR), and Akt signaling pathway. Specifically, MAP3K4 kinase inactivation in trophoblast stem (TS) cells results in reduced IGF1R and IR expression and decreased Akt activation. We observe these changes in TS cells also occur in differentiated trophoblasts created through in vitro differentiation of cultured TS cells and in vivo in placental tissues formed by TS cells. Furthermore, we show that MAP3K4 controls this pathway by promoting Igf1r transcript expression in TS cells through activation of CREB-binding protein (CBP). In the MAP3K4 kinase-inactive TS cells, Igf1r transcripts are repressed because of reduced CBP activity and increased histone deacetylase 6 expression and activity. Together, these data demonstrate a critical role for MAP3K4 in promoting fetal and placental growth by controlling the activity of the IGF1R/IR and Akt signaling pathway., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Systems biology analysis of intracellular signaling networks has tremendously expanded our understanding of normal and diseased cell behaviors and has revealed paths to finding proper therapeutic molecular targets. When it comes to neurons in the human brain, analysis of intraneuronal signaling networks provides invaluable information on learning, memory and cognition-related disorders, as well as potential therapeutic targets. However, neurons in the human brain form a highly complex neural network that, among its many roles, is also responsible for learning, memory formation and cognition. Given the impairment of these processes in mental and psychiatric disorders, one can envision that analyzing interneuronal processes, together with analyzing intraneuronal signaling networks, can result in a better understanding of the pathology and, subsequently, more effective target discovery. In this paper, a hybrid model is introduced, composed of the long-term potentiation (LTP) interneuronal process and an intraneuronal signaling network regulating CREB. LTP refers to an increased synaptic strength over a long period of time among neurons, typically induced upon occurring an activity that generates high-frequency stimulations (HFS) in the brain, and CREB is a transcription factor known to be highly involved in important functions of the cognitive and executive human brain such as learning and memory. The hybrid LTP-signaling model is analyzed using a proposed molecular fault diagnosis method. It allows to study the importance of various signaling molecules according to how much they affect an intercellular phenomenon when they are faulty, i.e. dysfunctional. This paper is intended to suggest another angle for understanding the pathology and therapeutic target discovery by classifying and ranking various intraneuronal signaling molecules based on how much their faulty behaviors affect an interneuronal process. Possible relations between the introduced hybrid analysis and the previous purely intracellular analysis are investigated in the paper as well., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pain comorbid with depression occurred frequently in clinical settings. This study aims to explore the molecular mechanism underlying antidepressant and analgetic effect of salvianolic acid B (SalB) in comorbid pain in depression induced by chronic restraint stress (CRS), which associates with GABAergic neuron activation in the amygdala and the ERK-CREB-BDNF signaling pathway. The differentially expressed genes related to comorbid pain in CRS-induced depression were screened through bioinformatics analysis. After CRS treatment for 3 weeks, depression-like behaviors were developed in GAD2-tdT mice. The retrograde tracer cholera toxin B subunit combined with retrograde tracer CTB-488 was injected into the parafascicular nucleus of thalamus to project GABAergic neurons to observe the labeling of neurons in the whole brain. After treatment with SalB and ERK-CREB-BDNF signaling pathway inhibitor, CRS mice showed a variety of depression-like behaviors, accompanied by enhanced activity of GABAergic neurons in the amygdala projecting to parafascicular nucleus of thalamus. BDNF underexpression occurred in the CRS mice. Overexpressed BDNF activated ERK-CREB-BDNF signaling pathway to alleviate comorbid pain in CRS-induced depression. After intraperitoneal injection of SalB, the depression-like behaviors and pain threshold in CRS mice were alleviated, the effects of which could be eliminated by ERK-CREB-BDNF signaling pathway antagonist. Collectively, SalB inhibits the excitation of GABAergic neurons in the amygdala and activates the ERK-CREB-BDNF signaling pathway through the parafascicular nucleus of thalamus, whereby alleviating comorbid pain in CRS-induced depression in mice., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cancer is one of the most common causes of death globally. Despite extensive research and considerable advances in cancer therapy, the fundamentals of the disease remain unclear. Understanding the key signaling mechanisms that cause cancer cell malignancy may help to uncover new pharmaco-targets. Cyclic adenosine monophosphate (cAMP) regulates various biological functions, including those in malignant cells. Understanding intracellular second messenger pathways is crucial for identifying downstream proteins involved in cancer growth and development. cAMP regulates cell signaling and a variety of physiological and pathological activities. There may be an impact on gene transcription from protein kinase A (PKA) as well as its downstream effectors, such as cAMP response element-binding protein (CREB). The position of CREB downstream of numerous growth signaling pathways implies its oncogenic potential in tumor cells. Tumor growth is associated with increased CREB expression and activation. PKA can be used as both an onco-drug target and a biomarker to find, identify, and stage tumors. Exploring cAMP effectors and their downstream pathways in cancer has become easier using exchange protein directly activated by cAMP (EPAC) modulators. This signaling system may inhibit or accelerate tumor growth depending on the tumor and its environment. As cAMP and its effectors are critical for cancer development, targeting them may be a useful cancer treatment strategy. Moreover, by reviewing the material from a distinct viewpoint, this review aims to give a knowledge of the impact of the cAMP signaling pathway and the related effectors on cancer incidence and development. These innovative insights seek to encourage the development of novel treatment techniques and new approaches.

Inflammatory pathways represented by TLR4/NF-κB (Toll-like receptor 4/Nuclear factor-κB) axis signaling are activated in the pathogenesis of endotoxin-induced myocardial dysfunction (EIMD). However, the underlying mechanism by which NF-κB coordinates with other transcriptional coactivators/corepressors to regulate the expression of proinflammatory cytokine genes remains unclear. We established an EIMD-mouse model by intraperitoneal injection of lipopolysaccharides (LPS), and we discovered that NCOA1 (nuclear receptor coactivator 1) assembled with CBP (CREB binding protein) and NF-κB subunits to form a transcriptional complex that specifically bound to promoters of proinflammatory cytokine genes to activate their expression. LPS treatment also inhibited DNMT1 (DNA methyltransferase 1) expression, thereby decreasing DNA methylation of a CpG island located on the promoter of NCOA1 and causing NCOA1 overexpression. Screening small molecules that abolished NCOA1-CBP interaction in a yeast system identified a compound PSSM2126 that effectively blocked the NCOA1-CBP interaction in vitro and in vivo. Administration of PSSM2126 to EIMD mice significantly alleviated the inflammation response and improved cardiac function. Collectively, our results reveal that an NCOA1-dependent transactivation mechanism can regulate proinflammatory cytokine expression, thereby improving our understanding of the activation of NF-κB targets. The promising inhibition of the NCOA1-CBP interaction by PSSM2126 may provide a new therapeutic option for EIMD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known., Methods: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300 iKO or CBP iKO ), and in samples of human intimal hyperplasia., Results: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300 iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBP iKO mice were entirely protected. In normal aorta, p300 iKO reduced, but CBP iKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia., Conclusions: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

The posttranslational regulation of the neuronal proteome is critical for brain homeostasis but becomes dysregulated in the aged or diseased brain, in which abnormal posttranslational modifications (PTMs) are frequently observed. While the full extent of modified substrates that comprise the "PTM-ome" are slowly emerging, how the upstream enzymes catalyzing these processes are regulated themselves is not well understood, particularly in the context of neurodegeneration. Here, we describe the reciprocal regulation of a kinase, the microtubule affinity-regulating kinase 2 (MARK2), and an acetyltransferase, CREB-binding protein (CBP), two enzymes known to extensively modify tau proteins in the progression of Alzheimer's disease. We found that MARK2 negatively regulates CBP and, conversely, CBP directly acetylates and inhibits MARK2 kinase activity. These findings highlight a reciprocal negative feedback loop between a kinase and an acetyltransferase, which has implications for how PTM interplay is coordinated on substrates including tau. Our study suggests that PTM profiles occur through the posttranslational control of the master PTM remodeling enzymes themselves., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The coactivator KIX of CBP uses two binding surfaces to recognize multiple activators and exhibits allostery in ternary complex formation. Activator•coactivator interactions are central to transcriptional regulation, yet the microscopic origins of allostery in dynamic proteins like KIX are largely unknown. Here, we investigate the molecular recognition and allosteric manifestations involved in two KIX ternary systems c-Myb•KIX•MLL and pKID•KIX•MLL. Exploring the hypothesis that binary complex formation prepays an entropic cost for positive cooperativity, we utilize molecular dynamics simulations, side chain methyl order parameters, and differential scanning fluorimetry (DSF) to explore conformational entropy changes in KIX. The protein's configurational micro-states from structural clustering highlight the utility of protein plasticity in molecular recognition and allostery. We find that apo KIX occupies a wide distribution of lowly-populated configurational states. Each binding partner has its own suite of KIX states that it selects, building a model of molecular recognition fingerprints. Allostery is maximized with MLL pre-binding, which corresponds to the observation of a significant reduction in KIX micro-states observed when MLL binds. With all binding partners, the changes in KIX conformational entropy arise predominantly from changes in the most flexible loop. Likewise, we find that a small molecule and mutations allosterically inhibit/enhance activator binding by tuning loop dynamics, suggesting that loop-targeting chemical probes could be developed to alter KIX•activator interactions. Experimentally capturing KIX stabilization is challenging, particularly because of the disordered nature of particular activators. However, DSF melting curves allow for inference of relative entropic changes that occur across complexes, which we compare to our computed entropy changes using simulation methyl order parameters., Competing Interests: The authors have declared that no competing interests exist.

Circadian genes in the medial accumbal shell (mNAcSh) region regulate binge alcohol consumption. Here, we investigated if antisense-induced knockdown of major circadian genes (Per1, Per2, and NPAS2) in the mNAcSh of mice exposed to intermittent access two-bottle choice (IA2BC) paradigm modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP), key epigenetic modifiers associated with withdrawal-associated behaviors such as anxiety. Adult male C57BL/6J mice (N = 28), surgically implanted with bilateral guide cannulas above the mNAcSh, were chronically (4 weeks) exposed to alcohol (20% v/v) or saccharin (0.03%) via IA2BC paradigm. In the fourth week, a mixture of antisense (AS-ODNs; N = 14/group) or nonsense (NS-ODNs; N = 14/group) oligodeoxynucleotides against circadian genes were bilaterally infused into the mNAcSh. Subsequently, alcohol/saccharin consumption and preference were measured followed by euthanization of animals and verification of microinjection sites by visual inspection and the expression of HDAC-2 and CBP by using RT-PCR along with the verification of antisense-induced downregulation of circadian genes in the mNAcSh. As compared with NS-ODNs, AS-ODNs infusion significantly attenuated the alcohol-induced increase in HDAC-2 and reduction in CBP expression in the mNAcSh along with a significant reduction in alcohol consumption and preference. No significant effect was observed on either saccharin consumption or preference. Our results suggest that circadian genes in the mNAcSh may have a causal to play in mediating epigenetic changes observed after chronic alcohol consumption., (© 2021 International Society for Neurochemistry.)

Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer., Competing Interests: Authors are current or former employees and stockholders of Constellation Pharmaceuticals, a Morphosys company, which provided funding for this research. Patents have been filed around the chemical series that includes CPI-1612. This work does not relate to any marketed products or products in development. These disclosures do not alter our adherence to PLOS ONE policies on sharing data and materials.

Bone marrow mesenchymal stem cells (BMSCs) can boost osteosarcoma (OS) cell proliferation and invasion, yet the function of extracellular vesicles (EVs) derived from BMSCs on OS is scarcely known. This study is aimed at examining the role of BMSC-EVs in OS cells. BMSCs and BMSC-EVs were isolated and identified. The effect of EVs and EVs-si-NORAD on OS cell proliferation, invasion, migration, and angiogenesis was determined. Expressions of NORAD, miR-877-3p, and CREBBP were detected. The binding relationship among NORAD, miR-877-3p, and CREBBP was verified. The miR-877-3p inhibitor or pc-CREBBP was delivered into OS cells treated with EVs-si-NORAD for in vitro analysis. The nude mouse model of the subcutaneous tumor xenograft was established for in vivo analysis. BMSC-EVs promoted OS cell proliferation, invasion, migration, and angiogenesis. BMSC-EVs carried NORAD into OS cells and upregulated CREBBP by sponging miR-877-3p. miR-877-3p downregulation or CREBBP overexpression partly inverted the inhibitory effect of EVs by silencing NORAD on OS cell proliferation, invasion, migration, and angiogenesis. In vivo experiments validated that BMSC-EV-derived NORAD facilitated tumor growth by upregulating CREBBP via miR-877-3p. To conclude, BMSC-EV-derived NORAD facilitated OS cell proliferation, invasion, migration, and angiogenesis by modulating CREBBP via miR-877-3p, which may offer new insights into OS treatment., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Dapeng Feng et al.)

The tight control of transcriptional coactivators is a fundamental aspect of gene expression in cells. The regulation of the CREB-binding protein (CBP) and p300 coactivators, two paralog multidomain proteins, involves an autoinhibitory loop (AIL) of the histone acetyltransferase (HAT) domain. There is experimental evidence for the AIL engaging with the HAT binding site, thus interrupting the acetylation of histone tails or other proteins. Both CBP and p300 contain a domain of about 110 residues (called the bromodomain) that recognizes histone tails with one or more acetylated lysine side chains. Here, we investigate by molecular dynamics simulations whether the AIL of CBP (residues 1556-1618) acetylated at the side chain of Lys1595 can bind to the bromodomain. The structural instability and fast unbinding kinetics of the AIL from the bromodomain pocket suggest that the AIL is not a ligand of the bromodomain on the same protein chain. This is further supported by the absence of strong and persistent contacts at the binding interface. Furthermore, the simulations of unbinding show an initial fast detachment of the acetylated lysine and a slower phase necessary for complete AIL dissociation. We provide further evidence for the instability of the AIL intramolecular binding by comparison with a natural ligand, the histone peptide H3K56ac, which shows higher stability in the pocket.

As an important hormone that regulates the balance of calcium and phosphorus, parathyroid hormone (PTH) has also been found to have an important function in intervertebral disc degeneration (IVDD). Our aim was to investigate the mechanism by which PTH alleviates IVDD. In this study, the PTH 1 receptor was found to be highly expressed in severely degenerated human nucleus pulposus (NP) cells. We found in the mouse model of IVDD that supplementation with exogenous PTH alleviated the narrowing of the intervertebral space and the degradation of the extracellular matrix (ECM) caused by tail suspension (TS). In addition, inflammation, oxidative stress, and apoptosis levels were significantly increased in the intervertebral disc tissues of TS-induced mice, and the activity of NP cells was decreased. TS also led to the downregulation of Sonic hedgehog (SHH) signalling pathway-related signal molecules in NP cells such as SHH, Smoothened, and GLI1. However, supplementation with exogenous PTH can reverse these changes. In vitro , PTH also promotes the activity of NP cells and the secretion of ECM. However, the antagonist of the SHH signalling pathway can inhibit the therapeutic effect of PTH on NP cells. In addition, a cAMP-response element-binding protein, as an important transcription factor, was found to mediate the promotion of PTH on the SHH signalling pathway. Our results revealed that PTH can alleviate IVDD by inhibiting inflammation, oxidative stress, and apoptosis and improving the activity of NP cells via activating the SHH signalling pathway., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 You Li et al.)

Serotonin transporter gene-linked polymorphic region polymorphisms are associated with anxiety, neuroticism, affective disorders and vulnerability to stressful life events; however, the relevant physiological mechanisms are not well understood. Serotonin transporter knockout mice have been widely used as a model of allelic variation of serotonin transporter function in humans; herein, wild-type mice and heterozygous and homozygous knockout mice models were established to explore the behavioural changes related to different genotypes and the possible physiological mechanisms. Behavioural changes were assessed using behavioural tests, namely, elevated plus maze, open field, Morris water maze and rotarod tests. Serum indicators were detected using the enzyme-linked immunosorbent assay. Compared with wild-type mice, homozygous mice showed significant anxiety-like behaviours in the plus maze and open field tests; conversely, anxiety-like behaviours in heterozygous mice were less pronounced. Homozygous mice also showed cognitive impairment and motor inhibition in the Morris water maze and rotarod tests. Serotonin levels decreased in both heterozygous and homozygous mice, and 5-hydroxytryptophan, protein kinase A, adenylyl cyclase, cyclic adenosine monophosphate response element-binding protein and brain-derived neurotrophic factor levels were lower in homozygous mice than in wild-type and heterozygous mice, whereas no statistical differences were found between wild-type and heterozygous mice. Additionally, there was a correlation between serological and behavioural indicators. This study provided experimental evidence that the cyclic adenosine monophosphate-protein kinase A-cyclic adenosine monophosphate response element-binding protein-brain-derived neurotrophic factor pathway may be involved in the regulation of polymorphism to stress and enriched the behavioural and physiological characteristics of serotonin transporter knockout mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rheumatoid arthritis (RA) is chronic inflammatory autoimmune disease. The crucial role of long non-coding RNA (lncRNA) in the progression of RA has been highlighted. Hence, this study was designed to explore the specific downstream mechanism of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in RA. Initially, the expression of NEAT1, p-p65, p300, and IL-18 in clinical tissues and cells was determined. Then, interactions among p65, NEAT1, p300, CBP, and IL-18 were investigated by immunofluorescence staining, dual luciferase reporter gene assay, RT-qPCR assay ChIP assay, and RIP assay followed by the analysis of their effects on RA in vivo and in vitro after expression alteration. The expressions of NEAT1, p-p65, p300, and IL-18 were all upregulated in the synovial tissues from the mice and patients with RA. NEAT1 silencing reduced the infiltration of CD4 + T cells and macrophages in synovial tissues, downregulated expression of blood inflammatory factors, relieved RA severity, and lowered incidence of RA in mice. Further, p-p65 could increase the expression of NEAT1 by binding to the NEAT1 promoter region, NEAT1 could co-locate and interact with p300, thus regulating the expression of IL-18 by regulating histone acetylation modification in IL-18 promoter region. NEAT1 aggravated RA via p300/CBP/IL-18 axis, representing a promising therapeutic target in RA., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Alzheimer's disease (AD) is a neurodegenerative disease that is characterized by loss of memory and cognitive impairment via dysfunction of the cholinergic nervous system. In cholinergic dysfunction, it is well known that impaired cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) signaling are major pathological markers and are some of the strategies for the development of AD therapy. Therefore, this study is aimed at evaluating whether a mixture comprising Ginkgo biloba L . leaf (GL) and Hericium erinaceus (Bull.) Pers . (HE) fruit extract (GH mixture) alleviated cognitive impairment induced in a scopolamine-induced model. It was discovered that GH reduced neuronal apoptosis and promoted neuronal survival by activating BDNF signaling in an in vitro assay. In addition, the GH ( p.o. 240 mg/kg) oral administration group significantly restored the cognitive deficits of the scopolamine-induced mouse group ( i.p. 1.2 mg/kg) in the behavior tests such as Y-maze and novel object recognition task (NORT) tests. This mixture also considerably enhanced cholinergic system function in the mouse brain. Furthermore, GH markedly upregulated the expressed levels of extracellular signal-regulated kinase (ERK), CREB, and BDNF protein levels. These results demonstrated that GH strongly exerted a neuroprotective effect on the scopolamine-induced mouse model, suggesting that an optimized mixture of GL and HE could be used as a good material for developing functional foods to aid in the prevention of neurodegenerative diseases, including AD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Seong Min Hong et al.)

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb-KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb-KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb-KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein-protein interactions., (© 2022. The Author(s).)

Circular RNA CREB-binding protein (circ-CREBBP) has been reported to involve in the tumorigenesis of glioma. However, the role and underlying molecular mechanism of circ-CREBBP in glioma glutamine catabolism remain unclear. The expression of circ-CREBBP, microRNA (miR)-375 and glutaminase (GLS) was detected using quantitative real-time polymerase chain reaction and western blot. The 3‑(4, 5‑dimethylthiazol‑2‑y1)‑2, 5‑diphenyl tetrazolium bromide (MTT), colony formation, flow cytometry and transwell assays were used to determine the effects of them on glioma cell malignant biological behaviors in vitro. Glutamine metabolism was analyzed using assay kits. Murine xenograft model was established to investigate the role of circ-CREBBP in vivo. The binding interactions between miR-375 and circ-CREBBP or GLS were confirmed by the dual-luciferase reporter assay. Circ-CREBBP was highly expressed in glioma tissues and cells, and high expression of circ-CREBBP predicted poor prognosis. Circ-CREBBP knockdown suppressed cell proliferation, migration, invasion and glutamine metabolism while expedited cell apoptosis in glioma in vitro, as well as impeded tumor growth in vivo. Circ-CREBBP directly targeted miR-375, which was demonstrated to restrain glioma cell growth, motility and glutamine metabolism. Moreover, miR-375 inhibition reverted the anticancer effects of circ-CREBBP knockdown on glioma cells. GLS was a target of miR-375, GLS silencing or the treatment of GLS inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) impaired glioma cell malignant phenotypes and glutamine metabolism. Importantly, GLS up-regulation weakened the tumor-suppressive functions of miR-375 on glioma cells. Mechanistically, circ-CREBBP indirectly regulated GLS expression through sponging miR-375. In all, circ-CREBBP expedited glioma tumorigenesis and glutamine metabolism through miR-375/GLS axis, suggesting a promising target for combined glioma therapy., (Copyright © 2021. Published by Elsevier B.V.)

Cancer is often called a disease of aging. There are numerous ways in which cancer epidemiology and behaviour change with the age of the patient. The molecular bases for these relationships remain largely underexplored. To characterise them, we analyse age-associations in the nuclear and mitochondrial somatic mutational landscape of 20,033 tumours across 35 tumour-types. Age influences both the number of mutations in a tumour (0.077 mutations per megabase per year) and their evolutionary timing. Specific mutational signatures are associated with age, reflecting differences in exogenous and endogenous oncogenic processes such as a greater influence of tobacco use in the tumours of younger patients, but higher activity of DNA damage repair signatures in those of older patients. We find that known cancer driver genes such as CDKN2A and CREBBP are mutated in age-associated frequencies, and these alter the transcriptome and predict for clinical outcomes. These effects are most striking in brain cancers where alterations like SUFU loss and ATRX mutation are age-dependent prognostic biomarkers. Using three cancer datasets, we show that age shapes the somatic mutational landscape of cancer, with clinical implications., (© 2022. The Author(s).)

The molecular targets and mechanisms of propolis ameliorating metabolic syndrome are not fully understood. Here, we report that Brazilian green propolis reduces fasting blood glucose levels in obese mice by disrupting the formation of CREB/CRTC2 transcriptional complex, a key regulator of hepatic gluconeogenesis. Using a mammalian two-hybrid system based on CREB-CRTC2, we identify artepillin C (APC) from propolis as an inhibitor of CREB-CRTC2 interaction. Without apparent toxicity, APC protects mice from high fat diet-induced obesity, decreases fasting glucose levels, enhances insulin sensitivity and reduces lipid levels in the serum and liver by suppressing CREB/CRTC2-mediated both gluconeogenic and SREBP transcriptions. To develop more potential drugs from APC, we designed and found a novel compound, A57 that exhibits higher inhibitory activity on CREB-CRTC2 association and better capability of improving insulin sensitivity in obese animals, as compared with APC. In this work, our results indicate that CREB/CRTC2 is a suitable target for developing anti-metabolic syndrome drugs., (© 2022. The Author(s).)

Nuclear-localized epidermal growth factor receptor (EGFR) highly correlates with the malignant progression and may be a promising therapeutic target for breast cancer. However, molecular mechanisms of nuclear EGFR in triple-negative breast cancer (TNBC) have not been fully elucidated. Here, we performed gene-annotation enrichment analysis for the interactors of nuclear EGFR and found that RNA-binding proteins (RBPs) were closely associated with nuclear EGFR. We further demonstrated p54 nrb /NONO, one of the RBPs, significantly interacted with nuclear EGFR. NONO was upregulated in 80 paired TNBC tissues and indicated a poor prognosis. Furthermore, NONO knockout significantly inhibited TNBC proliferation in vitro and in vivo. Mechanistically, NONO increased the stability of nuclear EGFR and recruited CREB binding protein (CBP) and its accompanying E1A binding protein p300, thereby enhancing the transcriptional activity of EGFR. In turn, EGFR positively regulated the affinity of NONO to mRNAs of nuclear EGFR downstream genes. Furthermore, the results indicated that the nuclear EGFR/NONO complex played a critical role in tumorigenesis and chemotherapy resistance. Taken together, our findings indicate that NONO enhances nuclear EGFR-mediated tumorigenesis and may be a potential therapeutic target for TNBC patients with nuclear EGFR expression., (© 2021. The Author(s).)

Gonadal function is connected to hypoxia, with hypoxia-inducible factor (HIF) 1α, as a component of HIF1-complexes, regulating cellular adaptation to hypoxic conditions. In the ovary, it regulates follicular maturation, ovulation and luteal development. At the cellular level, HIF1-complexes coordinate the expression of steroidogenic acute regulatory protein (STAR), and thereby ovarian steroidogenesis. The functionality of STAR is associated with the cAMP/PKA-dependent pathways. In vitro, HIF1α is required for basal and cAMP-induced STAR expression, under ambient and reduced oxygen (O 2 ) tension. Lowering O 2 increases the responsiveness of the Star promoter towards cAMP and PKA mediates activation/phosphorylation (P) of several transcriptional factors, including cJUN and cAMP response element-binding protein (CREB), whose functionality is linked to HIF1 through utilization of CREB-binding protein (CBP). Since the mechanisms underlying HIF1α-dependent expression of STAR remain unknown, we investigated the involvement of HIF1α in CREB-, cJUN- and CBP-mediated expression of STAR using a well-characterized steroidogenic model, murine KK1 granulosa cells; ambient and lowered (10%) O 2 were applied. Our main findings were that while functional suppression of the α-subunit of HIF1 lowered STAR/P-STAR and steroidogenic output from granulosa cells, surprisingly the levels of P-CREB and its transcriptional activity were strongly induced. However, its association with the Star promoter was decreased, indicating dissociation of P-CREB from the promoter. Further, suppression of HIF1 activity ultimately diminished the expression of cJUN/P-cJUN and CBP. Finally, the study suggests that HIF1-complex: (1) regulates cJUN expression in granulosa cells, (2) is involved in regulating the recruitment of P-CREB to the Star promoter in (3) a mechanism which possibly involves the HIF1-dependent regulation of CBP expression., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Dysregulated activation of the WNT/β-catenin signaling pathway is essential for the initiation and development of various cancers. E7386, a small-molecule compound, attenuates WNT signaling by blocking the interaction between β-catenin and CREB-binding protein (CBP); hence, it is regarded as a therapeutic candidate for cancers with activated WNT signaling. In the present study, we evaluated the biological characteristics associated with E7386 sensitivity by using a panel of patient-derived colon cancer spheroids. An integrative approach that combined E7386 sensitivity and gene expression profiles revealed that the resistance of the cancer spheroids to E7386 was associated with the activation of the NF-κB pathway. NF-κB pathway inhibitors acted synergistically with E7386 to block proliferation and induce cell cycle arrest in E7386-resistant spheroids. These findings suggest a possibility that a combination of E7386 and NF-κB inhibition may effectively block the proliferation of a subset of colon cancer cells., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Atsushi Ochiai reports financial support was provided by The Japan Agency for Medical Research and Development. Yusuke Kanda reports financial support was provided by Foundation for Promotion of Cancer Research., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Postoperative fatigue (POF) is the most common and long-lasting complication after surgery, which brings heavy burden to individuals and society. Recently, hastening postoperative recovery receives increasing attention, but unfortunately, the mechanisms underlying POF remain unclear. Propofol is a wildly used general anesthetic in clinic, and inspired by the rapid antidepressant effects induced by ketamine at non-anesthetic dose, the present study was undertaken to investigate the anti-fatigue effects and underlying mechanisms of propofol at a non-anesthetic dose in 70% hepatectomy induced POF model in rats. We first showed here that single administration of propofol at 0.1 mg/kg ameliorated acute POF in hepatectomy induced POF rats. Based on metabonomics analysis, we hypothesized that propofol exerted anti-fatigue activity in POF rats by facilitating free fatty acid (FFA) oxidation and gluconeogenesis. We further confirmed that propofol restored the deficit in FFA oxidation and gluconeogenesis in POF rats, as evidenced by the elevated FFA utilization, acetyl coenzyme A content, pyruvic acid content, phosphoenolpyruvic acid content, hepatic glucose output and glycogen storage. Moreover, propofol stimulated glucagon secretion and up-regulated expression of cAMP-response element binding protein (CREB), phosphorylated CREB, peroxlsome prolifeator-activated receptor-γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinade1 and carnitine palmitoltransferase 1A. In summary, our study suggests for the first time that propofol ameliorates acute POF by promoting glucagon-regulated gluconeogenesis via CREB/PGC-1α signaling and accelerating FFA beta-oxidation., Competing Interests: Declaration of competing interest The author have declared that no competing interest exists., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Infection of mammalian cells with viruses activates NF-κB to induce the expression of cytokines and chemokines and initiate an antiviral response. Here, we show that a vaccinia virus protein mimics the transactivation domain of the p65 subunit of NF-κB to inhibit selectively the expression of NF-κB-regulated genes. Using co-immunoprecipitation assays, we found that the vaccinia virus protein F14 associates with NF-κB co-activator CREB-binding protein (CBP) and disrupts the interaction between p65 and CBP. This abrogates CBP-mediated acetylation of p65, after which it reduces promoter recruitment of the transcriptional regulator BRD4 and diminishes stimulation of NF-κB-regulated genes CXCL10 and CCL2. Recruitment of BRD4 to the promoters of NFKBIA and CXCL8 remains unaffected by either F14 or JQ1 (a competitive inhibitor of BRD4 bromodomains), indicating that BRD4 recruitment is acetylation-independent. Unlike other viral proteins that are general antagonists of NF-κB, F14 is a selective inhibitor of NF-κB-dependent gene expression. An in vivo model of infection demonstrated that F14 promotes virulence. Molecular mimicry of NF-κB may be conserved because other orthopoxviruses, including variola, monkeypox and cowpox viruses, encode orthologues of F14., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Intestinal melatonin exerts diverse biological effects on the body. Our previous research showed that the abundance of the butyrate-producing bacteria, Roseburia , is positively related to the expression of colonic mucosal melatonin. However, the detailed relationship is unclear. Therefore, we aimed to explore whether Roseburia regulates intestinal melatonin and its underlying mechanisms. Male Sprague-Dawley germfree rats were orally administered with or without Roseburia hominis . R. hominis treatment significantly increased the intestinal melatonin level. The concentrations of propionate and butyrate in the intestinal contents were significantly elevated after gavage of R. hominis . Propionate or butyrate treatment increased melatonin, 5-hydroxytryptamine (5-HT), arylalkylamine N-acetyltransferase (AANAT), and phosphorylated cAMP-response element-binding protein (p-CREB) levels. When pretreated with telotristat ethyl, the inhibitor of tryptophan hydroxylase (TPH), or siRNA of Aanat , or 666-15, i.e., an inhibitor of CREB, propionate, or butyrate, could not promote melatonin production in the pheochromocytoma cell line BON-1. Metabolomics analysis showed that propionate and butyrate stimulation regulated levels of some metabolites and some metabolic pathways in BON-1 cell supernatants. In conclusion, propionate and butyrate, i.e., metabolites of R. hominis , can promote intestinal melatonin synthesis by increasing 5-HT levels and promoting p-CREB-mediated Aanat transcription, thereby offering a potential target for ameliorating intestinal diseases.

Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-β induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-β induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo . Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS -/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.

Thyroid hormone (TH) and thyroid hormone receptor (THR) regulate stem cell proliferation and differentiation during development, as well as during tissue renewal and repair in the adult. THR undergoes posttranslational modification by small ubiquitin-like modifier (SUMO). We generated the THRA (K283Q/K288R) -/- mouse model for in vivo studies and used human primary preadipocytes expressing the THRA sumoylation mutant (K283R/K288R) and isolated preadipocytes from mutant mice for in vitro studies. THRA mutant mice had reduced white adipose stores and reduced adipocyte cell diameter on a chow diet, compared to wild-type, and these differences were further enhanced after a high fat diet. Reduced preadipocyte proliferation in mutant mice, compared to wt, was shown after in vivo labeling of preadipocytes with EdU and in preadipocytes isolated from mice fat stores and studied in vitro. Mice with the desumoylated THRA had disruptions in cell cycle G 1 /S transition and this was associated with a reduction in the availability of cyclin D2 and cyclin-dependent kinase 2. The genes coding for cyclin D1, cyclin D2, cyclin-dependent kinase 2 and Culin3 are stimulated by cAMP Response Element Binding Protein (CREB) and contain CREB Response Elements (CREs) in their regulatory regions. We demonstrate, by Chromatin Immunoprecipitation (ChIP) assay, that in mice with the THRA K283Q/K288R mutant there was reduced CREB binding to the CRE. Mice with a THRA sumoylation mutant had reduced fat stores on chow and high fat diets and reduced adipocyte diameter., (© 2021. The Author(s).)

Facilitated dissociation provides a mechanism by which high-affinity complexes can be rapidly disassembled. The negative feedback regulator CITED2 efficiently downregulates the hypoxic response by displacing the hypoxia-inducible transcription factor HIF-1α from the TAZ1 domain of the transcriptional coactivators CREB-binding protein (CBP) and p300. Displacement occurs by a facilitated dissociation mechanism involving a transient ternary intermediate formed by binding of the intrinsically disordered CITED2 activation domain to the TAZ1:HIF-1α complex. The short lifetime of the intermediate precludes straightforward structural investigations. To obtain insights into the molecular determinants of facilitated dissociation, we model the ternary intermediate by generating a fusion peptide composed of the primary CITED2 and HIF-1α binding motifs. X-ray crystallographic and NMR studies of the fusion peptide complex reveal TAZ1-mediated negative cooperativity that results in nearly mutually exclusive binding of specific CITED2 and HIF-1α interaction motifs, providing molecular-level insights into the allosteric switch that terminates the hypoxic response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The discovery of psychobiotics has improved the therapeutic choices available for clinical mental disorders and shows promise for regulating mental health in people by combining the properties of food and medicine. A Pediococcus acidilactici strain CCFM6432 was previously isolated and its mood-regulating effect was investigated in this study. Viable bacteria were given to chronically stressed mice for five weeks, and then the behavioral, neurobiological, and gut microbial changes were determined. CCFM6432 significantly reduced stress-induced anxiety-like behaviors, mitigated hypothalamic-pituitary-adrenal (HPA) axis hyperactivity, and reversed the abnormal expression of hippocampal phosphorylated CREB and the c-Fos protein. In particular, CCFM6432 improved the gut microbial composition by inhibiting the over-proliferated pathogenic bacteria ( e.g. , Escherichia-shigella ) and promoting beneficial bacteria growth ( e.g. , Bifidobacterium ). Lactic acid, rather than bacteriocin, was further confirmed as the key compound that determined the antimicrobial activity of CCFM6432. Collectively, these results first proved the psychobiotic potential of the Pediococcus acidilactici strain. Ingestion of CCFM6432, or fermented food containing it, may facilitate mental health management in daily life, especially during the COVID-19 pandemic.

Lysine crotonylation (Kcr) is a newly identified protein translational modification and is involved in major biological processes including glycolysis, but its role in colorectal cancer (CRC) is unknown. Here, we found that the Kcr of α enolase (ENO1) was significantly elevated in human CRC tissues compared with the paratumoral tissues. CREB-binding protein (CBP) functioned as a crotonyltranferase of ENO1, and SIRT2 was involved in the decrotonylation of ENO1. Using quantitative mass spectrometry for crotonylomics analysis, we further found that K420 was the main Kcr site of ENO1 and ENO1 K420 Kcr promoted the growth, migration, and invasion of CRC cells in vitro by enhancing the activity of ENO1 and regulating the expression of tumor-associated genes. Our study reveals an important mechanism by which ENO1 regulates CRC through crotonylation., Competing Interests: Declaration of competing interest The authors declare there is no conflict of interests., (Copyright © 2021. Published by Elsevier Inc.)

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment., (© 2021. The Author(s).)

Super-enhancers (SE) are clusters of transcription enhancers that drive gene expression. SEs are typically characterized by high levels of acetylation of histone H3 lysine 27 (H3K27ac), which is catalyzed by the histone lysine acetyltransferase CREB binding protein (CBP). Cancer cells frequently acquire tumor-specific SEs at key oncogenes, such as MYC , which induce several hallmarks of cancer. BRD4 is recruited to SEs and consequently functions as an epigenetic reader to promote transcription of SE-marked genes in cancer cells. miRNAs can be potent candidates for nucleic acid therapeutics for cancer. We previously identified miR-766-5p as a miRNA that downregulated MYC expression and inhibited cancer cell growth in vitro . In this study, we show that miR-766-5p directly targets CBP and BRD4. Concurrent suppression of CBP and BRD4 cooperatively downregulated MYC expression in cancer cells but not in normal cells. Chromatin immunoprecipitation analysis revealed that miR-766-5p reduced levels of H3K27ac at MYC SEs via CBP suppression. Moreover, miR-766-5p suppressed expression of a BRD4-NUT fusion protein that drives NUT midline carcinoma. In vivo administration of miR-766-5p suppressed tumor growth in two xenograft models. Collectively, these data suggest that targeting SEs using miR-766-5p -based therapeutics may serve as an effective strategy for the treatment of MYC-driven cancers. SIGNIFICANCE: This study demonstrates that miR-766-5p targets CBP and BRD4, which can mitigate the protumorigenic consequences of SEs and oncogenic fusion proteins., (©2021 American Association for Cancer Research.)

The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability., (© 2021 Wiley Periodicals LLC.)

Introduction: We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption., Methods: To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24 h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining., Results: Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed., Conclusions: Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption., (© 2021 by the Research Society on Alcoholism.)

The function of the transcription factor, cAMP response element-binding protein (CREB), is activated through S133 phosphorylation by PKA and others. Regarding its inactivation, it is not well defined. cAMP response element-binding protein plays an essential role in promoting cell proliferation, neuronal survival and the synaptic plasticity associated with long-term memory. Our recent studies have shown that CREB is an important player in mediating stress response. Here, we have demonstrated that CREB regulates aging process through suppression of αB-crystallin and activation of the p300-p53-Bak/Bax signaling axis. First, we determined that two specific protein phosphatases, PP-1β and PP-2Aα, can inactivate CREB through S133 dephosphorylation. Subsequently, we demonstrated that cells expressing the S133A-CREB, a mutant mimicking constant dephosphorylation at S133, suppress CREB functions in aging control and stress response. Mechanistically, S133A-CREB not only significantly suppresses CREB control of αB-crystallin gene, but also represses CREB-mediated activation of p53 acetylation and downstream Bak/Bax genes. cAMP response element-binding protein suppression of αB-crystallin and its activation of p53 acetylation are major molecular events observed in human cataractous lenses of different age groups. Together, our results demonstrate that PP-1β and PP-2Aα modulate CREB functions in aging control and stress response through de-regulation of αB-crystallin gene and p300-p53-Bax/Bak signaling axis, which regulates human cataractogenesis in the aging lens., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Objective: Arterial restenosis is the pathological narrowing of arteries after endovascular procedures, and it is an adverse event that causes patients to experience recurrent occlusive symptoms. Following angioplasty, vascular smooth muscle cells (SMCs) change their phenotype, migrate, and proliferate, resulting in neointima formation, a hallmark of arterial restenosis. SIKs (salt-inducible kinases) are a subfamily of the AMP-activated protein kinase family that play a critical role in metabolic diseases including hepatic lipogenesis and glucose metabolism. Their role in vascular pathological remodeling, however, has not been explored. In this study, we aimed to understand the role and regulation of SIK3 in vascular SMC migration, proliferation, and neointima formation., Approach and Results: We observed that SIK3 expression was low in contractile aortic SMCs but high in proliferating SMCs. It was also highly induced by growth medium in vitro and in neointimal lesions in vivo. Inactivation of SIKs significantly attenuated vascular SMC proliferation and up-regulated p21CIP1 and p27KIP1. SIK inhibition also suppressed SMC migration and modulated actin polymerization. Importantly, we found that inhibition of SIKs reduced neointima formation and vascular inflammation in a femoral artery wire injury model. In mechanistic studies, we demonstrated that inactivation of SIKs mainly suppressed SMC proliferation by down-regulating AKT (protein kinase B) and PKA (protein kinase A)-CREB (cAMP response element-binding protein) signaling. CRTC3 (CREB-regulated transcriptional coactivator 3) signaling likely contributed to SIK inactivation-mediated antiproliferative effects., Conclusions: These findings suggest that SIK3 may play a critical role in regulating SMC proliferation, migration, and arterial restenosis. This study provides insights into SIK inhibition as a potential therapeutic strategy for treating restenosis in patients with peripheral arterial disease.

This study was designed to test whether Cynopterus sphinx distress calls influence olfactory learning and memory in conspecifics. Bats were exposed to distress calls/playbacks (PBs) of distress calls/modified calls and were then trained to novel odors. Bats exposed to distress calls/PBs made significantly fewer feeding attempts and bouts of PBs exposed to modified calls, which significantly induced the expression of c-Fos in the caudomedial neostriatum (NCM) and the amygdala compared to bats exposed to modified calls and trained controls. However, the expression of c-Fos in the hippocampus was not significantly different between the experimental groups. Further, protein phosphatase-1 (PP-1) expression was significantly lower, and the expression levels of E1A homologue of CREB-binding protein (CBP) (P300), brain-derived neurotrophic factor (BDNF) and its tyrosine kinase B1 (TrkB1) receptor were significantly higher in the hippocampus of control/bats exposed to modified calls compared to distress calls/PBs of distress call-exposed bats. Exposure to the call possibly alters the reciprocal interaction between the amygdala and the hippocampus, accordingly regulating the expression levels of PP1, P300 and BDNF and its receptor TrkB1 following training to the novel odor. Thus, the learning and memory consolidation processes were disrupted and showed fewer feeding attempts and bouts. This model may be helpful for understanding the contributions of stressful social communications to human disorders., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Virus-host interactions are complicated processes, and multiple cellular proteins promote or inhibit viral replication through different mechanisms. Recent progress has implicated circular RNAs (circRNAs) in cancer biology and progression; however, the role of circRNAs in viral infection remains largely unclear. Here, we detected 11,620 circRNAs in A549 cells and found that 411 of them were differentially expressed in influenza virus-infected A549 cells. We characterized a novel intronic circRNA, AIVR, that was upregulated in influenza virus-infected A549 cells and found that silencing of AIVR significantly promoted influenza virus replication in A549 cells. We further found that AIVR predominantly localizes in the cytoplasm and works as a microRNA (miRNA) sponge. One of the miRNAs absorbed by AIVR binds the mRNA of CREBBP, which is an important component of the large nucleoprotein complex interferon beta (IFN-β) enhanceosome that accelerates IFN-β production. AIVR overexpression significantly increased the mRNA and protein levels of IFN-β in the influenza virus-infected A549 cells. Therefore, the upregulation of AIVR is a cellular antiviral strategy, with AIVR exerting its antiviral effect by absorbing miRNA and promoting the expression of CREBBP to facilitate IFN-β production. Our study provides new insights into the roles of circRNAs in the cellular innate antiviral response. IMPORTANCE Circular RNAs (circRNAs) are new members of the long noncoding RNA families and have been identified in a variety of organisms, including plants, animals, and humans. Accumulating data indicate that circRNAs perform multiple functions in a variety of cellular processes associated with human diseases, such as Alzheimer's disease and cancer; however, the roles of circRNAs in virus infection have been largely uninvestigated. In this study, we investigated the cellular circRNA response upon influenza virus infection and found that 411 circRNAs were differentially expressed in the virus-infected cells. We identified a novel human intronic circRNA (we named AIVR) that antagonizes influenza virus replication. Upregulated circRNA AIVR absorbs an miRNA that binds the mRNA of CREBBP, leading to an increase in the cellular expression of CREBBP and then accelerating IFN-β production. This study advances the understanding of the roles of circRNAs in the cellular innate antiviral response.

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression. Many studies have described the conserved functions of CBP/p300 in stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model for the in vivo study of the molecular mechanisms underlying stem cell differentiation during regeneration. However, how this process is regulated genetically and epigenetically is not well-understood yet. We identified 5 distinct Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses revealed that Smed-cbp-2 appears to be essential for stem cell maintenance. On the other hand, the silencing of Smed-cbp-3 resulted in the growth of blastemas that were apparently normal, but remained largely unpigmented and undifferentiated. Smed-cbp-3 silencing also affected the differentiation of several cell lineages including neural, epidermal, digestive, and excretory cell types. Finally, we analysed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their functions in planarian stem cell biology. Our results indicate that planarian cbp genes play key roles in stem cell maintenance and differentiation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lung diseases, including asthma, pose a serious global health issue. Loss of mitochondrial function and decreased mitochondrial biogenesis play pivotal roles in the initiation and progression of chronic lung diseases. Thus, maintaining mitochondrial function and homeostasis is an important treatment goal. Zafirlukast is a CysLTR1 antagonist that is widely used as an adjuvant treatment for asthma. In the present study, we investigated the effects of zafirlukast in vitro using human bronchial epithelial cells (BECs). We performed measurements of oxygen consumption and bioenergetics and found that zafirlukast increased mitochondrial respiration and biogenesis in human BECs as evidenced by increased mitochondrial mass and mtDNA/nDNA. Through real-time PCR and western blot analysis, we found that zafirlukast significantly increased the expression of PGC-1α, NRF1, and TFAM at both the mRNA and protein levels. Finally, we determined that these effects are mediated through CREB signaling and that inhibition of CREB with its specific inhibitor H89 abolished the effects of zafirlukast described above. Thus, zafirlukast might have potential in enhancing mitochondrial function by promoting mitochondrial biogenesis in human bronchial epithelial cells through upregulating the expression of PGC-1α and activating the CREB pathway., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

CREBBP (CBP/KAT3A) and its paralogue EP300 (KAT3B) are lysine acetyltransferases (KATs) that are essential for human development. They each comprise 10 domains through which they interact with >400 proteins, making them important transcriptional co-activators and key nodes in the human protein-protein interactome. The bromodomains of CREBBP and EP300 enable the binding of acetylated lysine residues from histones and a number of other important proteins, including p53, p73, E2F, and GATA1. Here, we report a work to develop a high-affinity, small-molecule ligand for the CREBBP and EP300 bromodomains [(-)-OXFBD05] that shows >100-fold selectivity over a representative member of the BET bromodomains, BRD4(1). Cellular studies using this ligand demonstrate that the inhibition of the CREBBP/EP300 bromodomain in HCT116 colon cancer cells results in lowered levels of c-Myc and a reduction in H3K18 and H3K27 acetylation. In hypoxia (<0.1% O 2 ), the inhibition of the CREBBP/EP300 bromodomain results in the enhanced stabilization of HIF-1α.

Retinal Ganglion Cells (RGCs) lose their ability to grow axons during development. Adult RGCs thus fail to regenerate their axons after injury, leading to vision loss. To uncover mechanisms that promote regeneration of RGC axons, we identified transcription factors (TF) and open chromatin regions that are enriched in rat embryonic RGCs (high axon growth capacity) compared to postnatal RGCs (low axon growth capacity). We found that developmental stage-specific gene expression changes correlated with changes in promoter chromatin accessibility. Binding motifs for TFs such as CREB, CTCF, JUN and YY1 were enriched in the regions of the chromatin that were more accessible in embryonic RGCs. Proteomic analysis of purified rat RGC nuclei confirmed the expression of TFs with potential role in axon growth such as CREB, CTCF, YY1, and JUND. The CREB/ATF binding motif was widespread at the open chromatin region of known pro-regenerative TFs, supporting a role of CREB in regulating axon regeneration. Consistently, overexpression of CREB fused to the VP64 transactivation domain in mouse RGCs promoted axon regeneration after optic nerve injury. Our study provides a map of the chromatin accessibility during RGC development and highlights that TF associated with developmental axon growth can stimulate axon regeneration in mature RGC., (© 2021. The Author(s).)

Gamma-glutamyltransferase (GGT), a marker of liver disease, has been shown to be associated with increased risk of diabetes and relative insulin secretion deficiency. However, the mechanism of hepatic Ggt regulation has not been explored fully. In this study, we made a concerted effort to understand the mechanism by investigating the effects of acetylation of histones H3 and H4, and bindings of histone acetyltransferases, CREB binding protein (CBP) and p300, at the Ggt promoter on the regulation of the expression of Ggt gene in the livers of streptozotocin (STZ)-induced moderate hypoinsulinemia rat model. The rats treated with STZ showed remarkably higher serum GGT level and hepatic Ggt/GGT expression than the untreated control rats. Furthermore, the acetylation of histones H3 and H4, and the binding of CBP not p300 at the Ggt promoter regions were significantly higher in the livers of STZ rats than those of the control rats. These results suggest that an enhanced hepatic expression of Ggt is associated with increased acetylation of histones H3 and H4 and CBP binding at the Ggt promoter in STZ-induced moderate hypoinsulinemic rats.

Acute lung injury induced by ischemia-reperfusion (I/R)-associated pulmonary inflammation is associated with high rates of morbidity. Despite advances in the clinical management of lung disease, molecular therapeutic options for I/R-associated lung injury are limited. Zinc finger protein 36 (ZFP36) is an AU-rich element-binding protein that is known to suppress the inflammatory response. A ZFP36 binding site occurs in the 3' UTR of the cAMP-response element-binding protein (CREB) binding protein (CREBBP) gene, which is known to interact with apoptotic proteins to promote apoptosis. In this study, we investigate the involvement of ZFP36 and CREBBP on I/R-induced lung injury in vivo and in vitro. Intestinal ischemia/reperfusion (I/R) activates inflammatory responses, resulting in injury to different organs including the lung. Lung tissues from ZFP36-knockdown mice and mouse lung epithelial (MLE)-2 cells were subjected to either Intestinal I/R or hypoxia/reperfusion, respectively, and then analyzed by Western blotting, immunohistochemistry, and real-time PCR. Silico analyses, pull down and RIP assays were used to analyze the relationship between ZFP36 and CREBBP. ZFP36 deficiency upregulated CREBBP, enhanced I/R-induced lung injury, apoptosis, and inflammation, and increased I/R-induced lung fibrosis. In silico analyses indicated that ZFP36 was a strong negative regulator of CREBBP mRNA stability. Results of pull down and RIP assays confirmed that ZFP36 direct interacted with CREBBP mRNA. Our results indicated that ZFP36 can mediate the level of inflammation-associated lung damage following I/R via interactions with the CREBBP/p53/p21/Bax pathway. The downregulation of ZFP36 increased the level of fibrosis.

Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET-based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chemoresistance is a major obstacle to the treatment of triple-negative breast cancer (TNBC), which has a poor prognosis. Increasing evidence has demonstrated the essential role of cancer stem cells (CSCs) in the process of TNBC chemoresistance. However, the underlying mechanism remains unclear. In the present study, we report that block of proliferation 1 (BOP1) serves as a key regulator of chemoresistance in TNBC. BOP1 expression was significantly upregulated in chemoresistant TNBC tissues, and high expression of BOP1 correlated with shorter overall survival and relapse-free survival in patients with TNBC. BOP1 overexpression promoted, while BOP1 downregulation inhibited the drug resistance and CSC-like phenotype of TNBC cells in vitro and in vivo. Moreover, BOP1 activated Wnt/β-catenin signaling by increasing the recruitment of cyclic AMP response element-binding protein (CBP) to β-catenin, enhancing CBP-mediated acetylation of β-catenin, and increasing the transcription of downstream stemness-related genes CD133 and ALDH1A1. Notably, treating with the β-catenin/CBP inhibitor PRI-724 induced an enhancement of chemotherapeutic response of paclitaxel in BOP1-overexpressing TNBC cells. These findings indicate that BOP1 is involved in chemoresistance development and might serve as a prognostic marker and therapeutic target in TNBC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation., Competing Interests: The authors have declared that no competing interests exist.