P300 Levels Research Articles

PPARβ/δ attenuates hepatic fibrosis by reducing SMAD3 phosphorylation and p300 levels via AMPK in hepatic stellate cells

The role of peroxisome proliferator-activated receptor (PPAR)β/δ in hepatic fibrosis remains a subject of debate. Here, we examined the effects of a PPARβ/δ agonist on the pathogenesis of liver fibrosis and the activation of hepatic stellate cells (HSCs), the main effector cells in liver fibrosis, in response to the pro-fibrotic stimulus transforming growth factor-β (TGF-β). The PPARβ/δ agonist GW501516 completely prevented glucose intolerance and peripheral insulin resistance, blocked the accumulation of collagen in the liver, and attenuated the expression of inflammatory and fibrogenic genes in mice fed a choline-deficient high-fat diet (CD-HFD). The antifibrogenic effect of GW501516 observed in the livers CD-HFD-fed mice could occur through an action on HSCs since primary HSCs isolated from Ppard-/- mice showed increased mRNA levels of the profibrotic gene Col1a1. Moreover, PPARβ/δ activation abrogated TGF-β1-mediated cell migration (an indicator of cell activation) in LX-2 cells (immortalized activated human HSCs). Likewise, GW501516 attenuated the phosphorylation of the main downstream intracellular protein target of TGF-β1, suppressor of mothers against decapentaplegic (SMAD)3, as well as the levels of the SMAD3 co-activator p300 via the activation of AMP-activated protein kinase (AMPK) and the subsequent inhibition of extracellular signal-regulated kinase-1/2 (ERK1/2) in LX-2 cells. Overall, these findings uncover a new mechanism by which the activation of AMPK by a PPARβ/δ agonist reduces TGF-β1-mediated activation of HSCs and fibrosis via the reduction of both SMAD3 phosphorylation and p300 levels.

Upregulation of p300 in paclitaxel-resistant TNBC: implications for cell proliferation via the PCK1/AMPK axis.

To explore the role of p300 in the context of paclitaxel (PTX) resistance in triple-negative breast cancer (TNBC) cells, focusing on its interaction with the phosphoenolpyruvate carboxykinase 1 (PCK1)/adenosine monophosphate-activated protein kinase (AMPK) pathway. The expression of p300 and PCK1 at the messenger ribonucleic acid (mRNA) level was detected using a quantitative polymerase chain reaction. The GeneCards and GEPIA databases were used to investigate the relationship between p300 and PCK1. The MDA-MB-231/PTX cell line, known for its PTX resistance, was chosen to understand the specific role of p300 in such cells. The Lipofectamine™ 3000 reagent was used to transfer the p300 small interfering RNA and the overexpression of PCK1 plasmid into MDA-MB-231/PTX. The expression levels of p300, PCK1, 5'AMPK and phosphorylated AMPK (p-AMPK) were determined using the western blot test. In TNBC cancer tissue, the expression of p300 was increased compared with TNBC paracancerous tissue (P < 0.05). In the MDA-MB-231 cell line of TNBC, the expression of p300 was lower than in the PTX-resistant TNBC cells (MDA-MB-231/PTX) (P < 0.05). The PCK1 expression was decreased in the TNBC cancer tissue compared with TNBC paracancerous tissue, and the PCK1 expression was reduced in MDA-MB-231/PTX than in MDA-MB-231 (P < 0.05) indicating that PCK1 was involved in the resistance function. Additionally, p-AMPK was decreased in MDA-MB-231/PTX compared with MDA-MB-231 (P < 0.05). The adenosine triphosphate (ATP) level was also detected and was significantly lower in MDA-MB-231/PTX than in MDA-MB-231 (P < 0.05). Additionally, cell proliferation increased significantly in MDA-MB-231/PTX at 48 and 72 h (P < 0.05) suggesting that MDA-MB-231/PTX cells obtained the resistance function which was associated with AMPK and ATP level. When p300 was inhibited, p-AMPK and ATP levels elevated in MDA-MB-231/PTX (P < 0.05). When PCK1 was suppressed, the ATP consumption rate decreased, and cell proliferation increased (P < 0.05). However, there were no changes in p300. In MDA-MB-231/PTX, p300 can inhibit p-AMPK and ATP levels by inhibiting PCK1 expression. Our findings suggest that targeting p300 could modulate the PCK1/AMPK axis, offering a potential therapeutic avenue for overcoming PTX resistance in TNBC.

Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer.

Aberrant protein post-translational modification is a hallmark of malignant tumors. Lysine succinylation (Ksucc) plays a vital role in cell energy metabolism in various cancers. However, whether succinylation can be catalyzed by acetyltransferase p300 remains unclear. In this study, we unveiled that p300 is a "writer" for succinylation, and p300-mediated Ksucc promotes cell glycometabolism in lung adenocarcinoma (LUAD). Specifically, our succinylome data revealed that EP300 deficiency leads to the systemic reduction of Ksucc, and 79.55% of the p300-succinylated proteins were found in the cytoplasm, which were primarily enriched in the carbohydrate metabolism process. Interestingly, deleting EP300 led to a notable decrease in Ksucc levels on several glycolytic enzymes, especially Phosphoglycerate Kinase 1 (PGK1). Mutation of the succinylated site of PGK1 notably hindered cell glycolysis and lactic acid excretion. Metabolomics in vivo indicated that p300-caused metabolic reprogramming was mainly attributed to the altered carbohydrate metabolism. In addition, 89.35% of LUAD patients exhibited cytoplasmic localization of p300, with higher levels in tumor tissues than adjacent normal tissues. High levels of p300 correlated with advanced tumor stages and poor prognosis of LUAD patients. Briefly, we disclose the activity of p300 to catalyze succinylation, which contributes to cell glucose metabolic reprogramming and malignant progression of lung cancer.

Targeting Super-Enhancers Driving EVI1 Expression in Leukemia By Inhibition of p300/CBP

Introduction One of the most aggressive types of acute myeloid leukemia (AML) is caused by aberrant activation of EVI1 as a result of the hijacking of enhancers repositioned by structural rearrangements. One of these enhancers, the MYC-blood enhancer cluster ( MYC-BENC) (Bahr et al. 2018) activates EVI1 in AML patients with t(3;8). MYC-BENC consists of several enhancer modules, of which only one is essential for activating EVI1: deletion of this element abrogates MYC-BENC to EVI1 promoter interaction as well as transcription of EVI1 (Ottema et al. 2021). Approach Given the central role of acetyltransferases p300 and CBP in enhancer activation, in particular super-enhancers such as MYC-BENC, we wondered whether we could specifically inhibit MYC-BENC driven EVI1 transcription using p300/CBP inhibitors. We studied this in a previously reported t(3;8) K562 model (Ottema et al. 2021), in which we introduced GFP 3' of EVI1 as a readout for MYC-BENC-driven EVI1 expression. Results ChIP-seq in t(3;8) cells showed that p300 binds to the promoter of EVI1 and to MYC-BENC, whereas CBP only binds to MYC-BENC (Fig. A). Following rapid p300/CBP degradation (3 hours) with the heterobifunctional p300/CBP degrader dCBP-1 (Vannam et al. 2021), we found a severe reduction of nascent EVI1 (and MYC) transcripts (SLAM-seq), as well as of EVI1 protein levels, i.e. by EVI1-eGFP, intracellular anti-EVI1-PE staining (Fig. B) and western blot.. Importantly, p300/CBP degradation was accompanied by loss of H3K27ac at MYC-BENC, but not at the promoter of EVI1 (Fig. A). Thus, the activation of EVI1 in t(3;8) AML appears strongly dependent on the presence of p300/CBP at MYC-BENC super-enhancer. Importantly, in healthy HSPCs, we observed that EVI1 expression was not strongly downregulated upon p300/CBP degradation (Fig. B). These data suggest that MYC-BENC driven EVI1 transcription in AML can be targeted, while leaving EVI1 transcription in normal HSPCs unaffected. We next studied whether loss of enhancer activity was dependent on functional domains within p300/CBP using specific inhibitors of either the acetyltransferase domain (A485) or bromodomain (GNE781). Treatment of t(3;8) EVI1-GFP cells with single inhibitors had a mild effect on EVI1 expression, but simultaneous A485/GNE781 treatment showed strong synergy in reducing EVI1 levels (Fig. B). Dual inhibition also led to a profound loss of H3K27ac signal at the MYC-BENC locus, but not at the EVI1 promoter (Fig. A). Thus, both domains in CBP/p300 are required to activate EVI1 expression via the hijacked MYC-BENC enhancer. As with dCBP-1, we found that EVI1 expression levels in healthy HSPCs were not affected by A485 and GNE781 treatment (Fig. B). We hypothesized that A485/GNE781 treatment would only cause loss of p300/CBP activity, but p300 ChIP-sequencing showed that A485/GNE781 treatment caused strong loss of p300 binding at the enhancer while the total protein levels of p300 and CBP were not affected. p300 binding at the EVI1 promoter was not decreased by A485/GNE781 treatment. In line with this, we observed that genome-wide p300 binding at enhancers was reduced at 15939 out of 41160 (39%) putative enhancers upon A485/GNE781 exposure, whereas dual inhibitor treatment only affected p300 binding at 429 out of 12452 promoters (3.5%) (Fig. A), suggesting a high selectivity of p300 inhibitors for long distant regulatory elements. Conclusion In summary, EVI1 is sensitive to pharmacological p300/CBP inhibition when activated by the hijacked MYC-BENC enhancer, but not in normal HSPCs. Globally, the sensitivity of genomic regions to bromo- or acetyltransferase inhibition is highly locus-dependent and is mainly restricted to enhancers. Our findings demonstrate that aberrantly controlled expression of oncogenes by hijacked enhancers in leukemia can be pharmacologically targeted, pointing to a potential therapeutic avenue for patients who are refractory to current treatment regimens.

Recombinant Human HAPLN1 Mitigates Pulmonary Emphysema by Increasing TGF-β Receptor I and Sirtuins Levels in Human Alveolar Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2030. One of its components, emphysema, has been defined as a lung disease that irreversibly damages the lungs' alveoli. Treatment is currently unavailable for emphysema symptoms and complete cure of the disease. Hyaluronan (HA) and proteoglycan link protein 1 (HAPLN1), an HA-binding protein linking HA in the extracellular matrix to stabilize the proteoglycan structure, forms a bulky hydrogel-like aggregate. Studies on the biological role of the full-length HAPLN1, a simple structure-stabilizing protein, are limited. Here, we demonstrated for the first time that treating human alveolar epithelial type 2 cells with recombinant human HAPLN1 (rhHAPLN1) increased TGF-β receptor 1 (TGF-β RI) protein levels, but not TGF-β RII, in a CD44-dependent manner with concurrent enhancement of the phosphorylated Smad3 (p-Smad3), but not p-Smad2, upon TGF-β1 stimulation. Furthermore, rhHAPLN1 significantly increased sirtuins levels (i.e., SIRT1/2/6) without TGF-β1 and inhibited acetylated p300 levels that were increased by TGF-β1. rhHAPLN1 is crucial in regulating cellular senescence, including p53, p21, and p16, and inflammation markers such as p-NF-κB and Nrf2. Both senile emphysema mouse model induced via intraperitoneal rhHAPLN1 injections and porcine pancreatic elastase (PPE)-induced COPD mouse model generated via rhHAPLN1-containing aerosols inhalations showed a significantly potent efficacy in reducing alveolar spaces enlargement. Preclinical trials are underway to investigate the effects of inhaled rhHAPLN1-containing aerosols on several COPD animal models.

Unravelling the Role of P300 and TMPRSS2 in Prostate Cancer: A Literature Review.

Prostate cancer is one of the most common malignant diseases in men, and it contributes significantly to the increased mortality rate in men worldwide. This study aimed to review the roles of p300 and TMPRSS2 (transmembrane protease, serine 2) in the AR (androgen receptor) pathway as they are closely related to the development and progression of prostate cancer. This paper represents a library-based study conducted by selecting the most suitable, up-to-date scientific published articles from online journals. We focused on articles that use similar techniques, particularly those that use prostate cancer cell lines and immunohistochemical staining to study the molecular impact of p300 and TMPRSS2 in prostate cancer specimens. The TMPRSS2:ERG fusion is considered relevant to prostate cancer, but its association with the development and progression as well as its clinical significance have not been fully elucidated. On the other hand, high p300 levels in prostate cancer biopsies predict larger tumor volumes, extraprostatic extension of disease, and seminal vesicle involvement at prostatectomy, and may be associated with prostate cancer progression after surgery. The inhibition of p300 has been shown to reduce the proliferation of prostate cancer cells with TMPRSS2:ETS (E26 transformation-specific) fusions, and combining p300 inhibitors with other targeted therapies may increase their efficacy. Overall, the interplay between the p300 and TMPRSS2 pathways is an active area of research.

Histone acetyltransferase P300 deficiency promotes ferroptosis of vascular smooth muscle cells by activating the HIF-1α/HMOX1 axis

BackgroundE1A-associated 300-kDa protein (P300), an endogenous histone acetyltransferase, contributes to modifications of the chromatin landscape of genes involved in multiple cardiovascular diseases. Ferroptosis of vascular smooth muscle cells (VSMCs) is a novel pathological mechanism of aortic dissection. However, whether P300 regulates VSMC ferroptosis remains unknown.MethodsCystine deprivation (CD) and imidazole ketone erastin (IKE) were used to induce VSMC ferroptosis. Two different knockdown plasmids targeting P300 and A-485 (a specific inhibitor of P300) were used to investigate the function of P300 in the ferroptosis of human aortic smooth muscle cells (HASMCs). Cell counting kit-8, lactate dehydrogenase and flow cytometry with propidium iodide staining were performed to assess the cell viability and death under the treatment of CD and IKE. BODIPY-C11 assay, immunofluorescence staining of 4-hydroxynonenal and malondialdehyde assay were conducted to detect the level of lipid peroxidation. Furthermore, co-immunoprecipitation was utilized to explore the interaction between P300 and HIF-1α, HIF-1α and P53.ResultsCompared with normal control, the protein level of P300 was significantly decreased in HASMCs treated with CD and IKE, which was largely nullified by the ferroptosis inhibitor ferrostatin-1 but not by the autophagy inhibitor or apoptosis inhibitor. Knockdown of P300 by short-hairpin RNA or inhibition of P300 activity by A-485 promoted CD- and IKE-induced HASMC ferroptosis, as evidenced by a reduction in cell viability and aggravation of lipid peroxidation of HASMCs. Furthermore, we found that hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase 1 (HMOX1) pathway was responsible for the impacts of P300 on ferroptosis of HASMCs. The results of co-immunoprecipitation demonstrated that P300 and P53 competitively bound HIF-1α to regulate the expression of HMOX1. Under normal conditions, P300 interacted with HIF-1α to inhibit HMOX1 expression, while reduced expression of P300 induced by ferroptosis inducers would favor HIF-1α binding to P53 to trigger HMOX1 overexpression. Furthermore, the aggravated effects of P300 knockdown on HASMC ferroptosis were largely nullified by HIF-1α knockdown or the HIF-1α inhibitor BAY87-2243.ConclusionThus, our results revealed that P300 deficiency or inactivation facilitated CD- and IKE-induced VSMC ferroptosis by activating the HIF-1α/HMOX1 axis, which may contribute to the development of diseases related to VSMC ferroptosis.

Medial prefrontal cortical PPM1F alters depression-related behaviors by modifying p300 activity via the AMPK signaling pathway.

Protein phosphatase Mg2+/Mn2+-dependent 1F (PPM1F) is a serine/threonine phosphatase, and its dysfunction in depression in the hippocampal dentate gyrus has been previously identified. Nevertheless, its role in depression of another critical emotion-controlling brain region, the medial prefrontal cortex (mPFC), remains unclear. We explored the functional relevance of PPM1F in the pathogenesis of depression. The gene expression levels and colocalization of PPM1F in the mPFC of depressed mice were measured by real-time PCR, western blot and immunohistochemistry. An adeno-associated virus strategy was applied to determine the impact of knockdown or overexpression of PPM1F in the excitatory neurons on depression-related behaviors under basal and stress conditions in both male and female mice. The neuronal excitability, expression of p300 and AMPK phosphorylation levels in the mPFC after knockdown of PPM1F were measured by electrophysiological recordings, real-time PCR and western blot. The depression-related behavior induced by PPM1F knockdown after AMPKα2 knockout or the antidepressant activity of PPM1F overexpression after inhibiting acetylation activity of p300 was evaluated. Our results indicate that the expression levels of PPM1F were largely decreased in the mPFC of mice exposed to chronic unpredictable stress (CUS). Behavioral alterations relevant to depression emerged with short hairpin RNA (shRNA)-mediated genetic knockdown of PPM1F in the mPFC, while overexpression of PPM1F produced antidepressant activity and ameliorated behavioral responses to stress in CUS-exposed mice. Molecularly, PPM1F knockdown decreased the excitability of pyramidal neurons in the mPFC, and restoring this low excitability decreased the depression-related behaviors induced by PPM1F knockdown. PPM1F knockdown reduced the expression of CREB-binding protein (CBP)/E1A-associated protein (p300), a histone acetyltransferase (HAT), and induced hyperphosphorylation of AMPK, resulting in microglial activation and upregulation of proinflammatory cytokines. Conditional knockout of AMPK revealed an antidepressant phenotype, which can also block depression-related behaviors induced by PPM1F knockdown. Furthermore, inhibiting the acetylase activity of p300 abolished the beneficial effects of PPM1F elevation on CUS-induced depressive behaviors. Our findings demonstrate that PPM1F in the mPFC modulates depression-related behavioral responses by regulating the function of p300 via the AMPK signaling pathway.

Abstract B051: Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC

Abstract Background: Histone modifying enzymes (HMEs) hold significant promise as targets for drug discovery in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) dictate the presence of acetyl marks, which regulate the openness of chromatin and transcriptional activation. We previously developed a novel histone array on a chip to measure HME dysregulation with the development of castration resistance (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC. This alteration was subsequently identified in a subset of castration-resistant tumors (BMC Cancer 2017). The goal of the current study was to determine if the isolation of circulating tumor cells (CTCs) could be used as a biomarker to identify patients with altered p300 acetylation activity. Methods: Peripheral blood was collected from 13 patients (15 samples) with advanced PC after informed written consent under the University of Wisconsin IRB-approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using pre-conjugated, anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled Sera-Mag streptavidin-coated magnetic beads (SM). Cells bound to SMs were isolated with ESP technology using an automated magnetic PipetMax pipetting robot. SM-bound cells were moved through staining and washing wells prior to imaging at 20x with a Nikon Eclipse Ti fluorescent microscope and NIS-Elements AR 4.10 software, and finally images were analyzed with NIS Elements software. Results: We first optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in prostate cancer cell lines. Employing a selective inhibitor of p300 acetylation (A-485) reduced levels of p300 and H3K18 acetylation. We then confirmed that CTCs from patient samples were successfully captured by the EpCAM antibody and show clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Increased acetylation of p300 and H3K18 and decreased SIRT2 is found in 6/10 CRPC samples compared to HSPC patients. Paired samples collected from one patient show a marked increase in p300 activity, H3K18 hyperacetylation, and a decrease in SIRT2 expression in the later developed CRPC sample compared to the HSPC sample. Another CRPC subject, decreased acetyl p300 and histone acetyl-H3k18, and increased SIRT2 staining is noted several months after beginning enzalutamide administrated. Analysis in CTCs indicates a positive correlation between acetyl-p300 and H3K18ac (R=0.61) and between SIRT2 and H3K18ac (R=-0.59). Conclusions: This study suggests that acetyl-p300, H3K18ac, and SIRT2 are epigenetic biomarkers that increase in a subset of CTC from CRPC. These biomarkers could potentially be used for monitoring outcomes and predicting tumor responses to developing p300 acetylation inhibitors. Citation Format: Mikolaj Filon, Bing Yang, Jennifer Schehr, Anupama Singh, Marcelo Bigarella, John Denu, Joshua Lang, David Jarrard. Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B051.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( &gt; 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

Клинические и нейрофизиологические корреляты когнитивных нарушений у пациентов с атеросклеротическим поражением сонных артерий

INTRODUCTION: Cognitive disorders often do not come to the fore in the interpretation of clinical manifestations in hemodynamically significant stenoses of brachiocephalic arteries. In this regard, the objectification of cognitive disorders based on a battery of tests, as well as of their neurophysiological correlates, is an important task of both angiology and neurology. Endogenous evoked potential P300 is a frequently used neurophysiological phenomenon characterizing the psychophysiological functions of perception, attention, and thinking. AIM: To study the dynamics of cognitive functions and their neurophysiological correlates in patients with hemodynamically significant carotid artery stenosis before and after carotid endarterectomy. MATERIALS AND METHODS: An open prospective study included 60 patients divided to two groups. Group A included 30 ‘symptomatic’ patients with a hemodynamically significant lesion (stenosis) of the internal carotid artery and a history of acute cerebrovascular accident. Group B included 30 ‘asymptomatic patients’ without a history of cerebrovascular events. Within the study, before surgery for carotid stenosis and 6 months after, the following parameters were assessed: cognitive status (according to the MMSE, FAB, MoCA, NIHSS scales), and also clinical and neurophysiological correlates of cognitive functions (P300 evoked potential). RESULTS: In the group of patients with neurological disorders (group A), a reliably greater number of errors (p = 0.048), as well as a decrease in the amplitude (p = 0.006) and increase in latency (p = 0.022) of P2 component of P300 cognitive evoked potential were determined. Based on the parameters of P300 event-related potential, a logit regression model was created that permitted to classify patients to the above-mentioned groups. The cluster analysis of dynamics of cognitive functions was additionally performed before and after surgery. In cluster 1, there were 45% of symptomatic patients, 55% of asymptomatic ones, in cluster 2 — 58% and 32%, respectively, no reliable differences were found (chi-square 0.58; p = 0.445). Cluster 1 was characterized by initially higher level of test performance with some increase in this parameter after surgery; Cluster 2 was characterized by a lower level of these parameters, with the greatest differences found in frontal assessment battery (FAB) characteristics. Reliable differences in the level of P300 were found only after surgery: the greatest differences were recorded in the amplitude of P2, P3, N2 components. CONCLUSION: The factor of cognitive impairment is a separate phenomenon, not closely related to focal neurological syndrome in patients with carotid atherosclerosis. An objective neurophysiological criterion for cognitive impairment in these patients is the endogenous evoked potential P300. With this, the greatest differences between the neurophysiological correlates of cognitive disorders are determined 6 months after the operation for carotid stenosis.

Sodium butyrate inhibits high glucose-induced inflammation by controlling the acetylation of NF-κB p65 in human monocytes.

Hyperglycemia is a major cause of diabetes and diabetes-related diseases. Sodium butyrate (NaB) is a short-chain fatty acid derivative that produces dietary fiber by anaerobic bacterial fermentation in the large intestine and occurs in foods, such as Parmesan cheese and butter. Butyrate has been shown to prevent obesity, improve insulin sensitivity, and ameliorate dyslipidemia in diet-induced obese mice. Therefore, this study examined the effects and mechanism of NaB on the secretion of inflammatory cytokines induced by high glucose (HG) in THP-1 cells. THP-1 cells were used as an in vitro model for HG-induced inflammation. The cells were cultured under normal glycemic or hyperglycemic conditions with or without NaB (0-25 μM). Western blotting and quantitative polymerase chain reaction were used to evaluate the protein and mRNA levels of nuclear factor-κB (NF-κB), interleukin-6, tumor necrosis factor-α, acetylated p65, acetyl CREB-binding protein/p300 (CBP/p300), and p300 using THP-1 cells. Histone acetyltransferase (HAT), histone deacetylase (HDAC), and pro-inflammatory cytokine secretion activity were analyzed using an enzyme-linked immunosorbent assay. HG significantly upregulated histone acetylation, acetylation levels of p300, NF-κB activation, and inflammatory cytokine release in THP-1 cells. Conversely, the NaB treatment reduced cytokine release and NF-κB activation in HG-treated cells. It also significantly reduced p65 acetylation, CBP/p300 HAT activity, and CBP/p300 gene expression. In addition, NaB decreased the interaction of p300 in acetylated NF-κB and TNF-α. These results suggest that NaB suppresses HG-induced inflammatory cytokine production through HAT/HDAC regulation in monocytes. NaB has the potential for preventing and treating diabetes and its related complications.

Acetyltransferase p300 regulates atrial fibroblast senescence and age-related atrial fibrosis through p53/Smad3 axis.

Atrial fibrosis induced by aging is one of the main causes of atrial fibrillation (AF), but the potential molecular mechanism is not clear. Acetyltransferase p300 participates in the cellular senescence and fibrosis, which might be involved in the age-related atrial fibrosis. Four microarray datasets generated from atrial tissue of AF patients and sinus rhythm (SR) controls were analyzed to find the possible relationship of p300 (EP300) with senescence and fibrosis. And then, biochemical assays and in vivo electrophysiological examination were performed on older AF patients, aging mice, and senescent atrial fibroblasts. The results showed that (1) the left atrial tissues of older AF patients, aging mouse, and senescence human atrial fibroblasts had more severe atrial fibrosis and higher protein expression levels of p300, p53/acetylated p53 (ac-p53)/p21, Smad3/p-Smads, and fibrosis-related factors. (2) p300 inhibitor curcumin and p300 knockdown treated aging mouse and senescence human atrial fibroblasts reduced the senescence ratio of atrial fibroblasts, ameliorated the atrial fibrosis, and decreased the AF inducibility. In contrast, over-expression of p300 can lead to the senescence of atrial fibroblasts and atrial fibrosis. (3) p53 knockdown decreased the expression of aging and fibrosis-related proteins. (4) Co-immunoprecipitation and immunofluorescence showed that p53 forms a complex with smad3 and directly regulates the expression of smad3 in atrial fibroblasts. Our findings suggest that the mechanism of atrial fibrosis induced by aging is, at least, partially dependent on the regulation of p300, which provides new sights into the AF treatment, especially for the elderly.

A mink (Neovison vison) model of self-injury: Effects of CBP-CREB axis on neuronal damage and behavior.

Self-injurious behavior (SIB) is a clinically challenging problem in the general population and several clinical disorders. However, the precise molecular mechanism of SIB is still not clear. In this paper, the systematic investigation of the genesis and development of SIB is conducted based on behavioral and pathophysiology studies in mink (Neovison vison) models. The night-vision video was used to observe the mink behavior, and the duration was a month. HE stain was performed to characterize the pathology change in the brain of a mink. IHC assay was performed to conduct the protein level detection of Iba-1, p-CREB, CBP, and p300 in the brain tissues. Elisa assay was used to examine the levels of NfL and NfH in serum and CSF of mink. The qRT-PCR assay was used to detect the expression of Bcl-2, NOR1, FoxO4, c-FOS, CBP, and p300 in brain tissues. Western blot was used to detect the protein levels of p-CREB, CBP, and p300 in brain tissues. We also used Evans Blue as a tracer to detect whether the blood-brain barrier was impaired in the brain of mink. The behavioral test, histopathological and molecular biology experiments were combined in this paper, and the results showed that CBP was related to SIB. Mechanism analysis showed that the dysregulation of CBP in brain-activated CREB signaling will result in nerve damage of the brain and SIB symptoms in minks. More importantly, the CBP-CREB interaction inhibitor might help relieve SIB and nerve damage in brain tissues. Our results illustrate that the induction of CBP and the activation of CREB are novel mechanisms in the genesis of SIB. This finding indicates that the CBP-CREB axis is critical for SIB and demonstrates the efficacy of the CBP-CREB interaction inhibitor in treating these behaviors.

A rapid, non-invasive method for fatigue detection based on voice information.

Fatigue results from a series of physiological and psychological changes due to continuous energy consumption. It can affect the physiological states of operators, thereby reducing their labor capacity. Fatigue can also reduce efficiency and, in serious cases, cause severe accidents. In addition, it can trigger pathological-related changes. By establishing appropriate methods to closely monitor the fatigue status of personnel and relieve the fatigue on time, operation-related injuries can be reduced. Existing fatigue detection methods mostly include subjective methods, such as fatigue scales, or those involving the use of professional instruments, which are more demanding for operators and cannot detect fatigue levels in real time. Speech contains information that can be used as acoustic biomarkers to monitor physiological and psychological statuses. In this study, we constructed a fatigue model based on the method of sleep deprivation by collecting various physiological indexes, such as P300 and glucocorticoid level in saliva, as well as fatigue questionnaires filled by 15 participants under different fatigue procedures and graded the fatigue levels accordingly. We then extracted the speech features at different instances and constructed a model to match the speech features and the degree of fatigue using a machine learning algorithm. Thus, we established a method to rapidly judge the degree of fatigue based on speech. The accuracy of the judgment based on unitary voice could reach 94%, whereas that based on long speech could reach 81%. Our fatigue detection method based on acoustic information can easily and rapidly determine the fatigue levels of the participants. This method can operate in real time and is non-invasive and efficient. Moreover, it can be combined with the advantages of information technology and big data to expand its applicability.

In Vitro Assessment of the Gene Expression of EZH-2 and P300 During Motor Neuron Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells.

Maintenance of neurogenesis depends on the function of some histone-modifying enzymes; including Enhancer of zeste homolog 2 (EZH2) and histone acetyltransferases (P300). The mechanism of epigenetic regulation and gene expression underlying the transition of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) into MNs has not been fully clarified. Two morphogens; sonic hedgehog (Shh: 100 ng/mL) and retinoic acid (RA: 0.01 mM) were involved in the specification of hUCB-MSCs into MNs after MSC characterization using Flow cytometry. Real time-quantitative PCR and immunocytochemistry were performed to find the expression of the genes at the level of mRNA and protein. The expression of MN-related markers was confirmed at the level of mRNA and protein by induction of differentiation. The results were confirmed by immunocytochemistry and showed those mean cell percentages of 55.33%±15.885% and 49.67%±13.796% could express Islet-1 and ChAT, respectively. The gene expression level of Islet-1 and ChAT was significantly increased in the first and second week of exposure, respectively. After two weeks, the expression level of P300 and EZH-2 genes increased remarkably. No significant expression of Mnx-1 was detected when compared to the control sample. MN-related markers, Islet-1 and ChAT, were detected in differentiated cells of hUCB-MSCs, supporting the potency of cord blood cells in the regeneration of MN-related disorders. Assessing these epigenetic regulatory genes at the protein level can be suggested to confirm their functional epigenetic modifying effects during motor neuron differentiation.

Coactivator p300-induced H3K27 acetylation mediates lipopolysaccharide-induced inflammatory mediator synthesis

To investigate the role of acetylated modification induced by coactivator p300 in lipopolysaccharide (LPS)- induced inflammatory mediator synthesis and its molecular mechanism. Agilent SurePrint G3 Mouse Gene Expression V2 microarray chip and Western blotting were used to screen the molecules whose expression levels in mouse macrophages (RAW246.7) were correlated with the stimulation intensity of LPS. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (chip-qPCR) were used to verify the binding of the molecules to the promoters of IL-6 and TNF-α genes. The effects of transfection of RAW246.7 cells with overexpression or interfering plasmids on IL-6 and TNF-α synthesis were evaluated with ELISA, and the binding level of the target molecules and acetylation level of H3K27 in the promoter region of IL-6 and TNF-α genes were analyzed by chromatin immunoprecipitation sequencing technique (chip-seq). Gene microarray chip data and Western blotting both confirmed a strong correlation of p300 expression with the stimulation intensity of LPS. Immunocoprecipitation confirmed the binding between p300 and c-myb. The results of EMSA demonstrated that c-myb (P < 0.05), but not p300, could directly bind to the promoter region of IL-6 and TNF-α genes; p300 could bind to the promoters only in the presence of c-myb (P < 0.05). The expressions of p65, p300 and c-myb did not show interactions. Both p300 overexpression and LPS stimulation could increase the level of promoter-binding p300 and H3K27 acetylation level, thus promoting p65 binding and inflammatory gene transcription; such effects were obviously suppressed by interference of c-myb expression (P < 0.05). Interference of p65 resulted in inhibition of p65 binding to the promoters and gene transcription (P < 0.05) without affecting p300 binding or H3K27 acetylation level. LPS can stimulate the synthesis of p300, whose binding to the promoter region of inflammatory genes via c-myb facilitates the cohesion of p65 by inducing H3K27 acetylation, thus promoting the expression of the inflammatory genes.

The P300 acetyltransferase inhibitor C646 promotes membrane translocation of insulin receptor protein substrate and interaction with the insulin receptor

Inhibition of P300 acetyltransferase activity by specific inhibitor C646 has been shown to improve insulin signaling. However, the underlying molecular mechanism of this improvement remains unclear. In this study, we analyzed P300 levels of obese patients and found that they were significantly increased in liver hepatocytes. In addition, large amounts of P300 appeared in the cytoplasm. Inhibition of P300 acetyltransferase activity by C646 drastically increased tyrosine phosphorylation of the insulin receptor protein substrates (IRS1/2) without affecting the tyrosine phosphorylation of the beta subunit of the insulin receptor (IRβ) in hepatocytes in the absence of insulin. Since IRS1/2 requires membrane translocation and binding to inositol compounds for normal functions, we also examined the role of acetylation on binding to phosphatidylinositol(4,5)P2 and found that IRS1/2 acetylation by P300 reduced this binding. In contrast, we show that inhibition of IRS1/2 acetylation by C646 facilitates IRS1/2 membrane translocation. Intriguingly, we demonstrate that C646 activates IRβ′s tyrosine kinase activity and directly promotes IRβ interaction with IRS1/2, leading to the tyrosine phosphorylation of IRS1/2 and subsequent activation of insulin signaling even in the absence of insulin. In conclusion, these data reveal the unique effects of C646 in activating insulin signaling in patients with obesity and diabetes.

Ketogenic diet ameliorates lipid dysregulation in type 2 diabetic mice by downregulating hepatic pescadillo 1

BackgroundPrevious reports implied a possible link between PES1 and lipid metabolism. However, the role of PES1 in regulating T2DM related lipid metabolism and the effect of ketogenic diet (KD) on PES1 have not been reported. The aim of present study is to explore the role of PES1 in effects of KD on diabetic mice and its mediated mechanism.MethodsMale C57BL/6J and KKAy mice were fed with standard diet (SD) and KD, respectively. Simultaneously, McArdle 7777 cells were treated by β-hydroxybutyric acid (β-HB), Pes1 siRNA or Pes1 overexpression plasmid, respectively. Additionally, liver-conditional knockout (CKO) of Pes1 in vivo was applied.ResultsHepatic PES1 expression in diabetic mice was markedly increased, which was suppressed by KD feeding with an accompanying reduction of hepatic and plasma triglycerides (TG). In mice with CKO of Pes1, the protein levels of p300, SREBP1c, FASN, SCD1, Caspase1, NLRP3 and GSDMD were dramatically downregulated in livers, and the plasma and hepatic TG, IL-1β and IL-18 were decreased as well. The similar outcomes were also observed in β-HB and Pes1 knockdown treated hepatocytes. By contrast, Pes1 overexpression in cultured hepatocytes showed that these levels were significantly enhanced, which were, however reduced under β-HB treatment. Mechanistically, we discovered that β-HB decreased CHOP binding to the Pes1 promoters, resulting in the downregulation of PES1, thereby reducing PES1 binding to p300 and Caspase1 promoters. The inhibition of p300 and Caspase1 expression elicited the dramatic suppression of acetylation of SREBP1c via its interaction with p300, and the decreased GSDMD levels. Besides, knockdown of Caspase1 also alleviated the TG levels in cultured hepatocytes.ConclusionKD may improve lipid dysregulation in type 2 diabetic mice by downregulating hepatic PES1 expression.

Steroid Receptor Coregulators Can Modulate the Action of Progesterone Receptor during the Estrous Cycle in Cow Endometrium.

Simple SummaryProper functioning of the endometrium is necessary for the implantation of the embryo after fertilization and its development throughout pregnancy. The key role in this process plays appropriate action of progesterone through the nuclear receptor isoforms. The action of the receptor is regulated by the attachment of receptor modulators called coregulators which include coactivators and corepressors. Their improper expression in humans causes a malfunction of progesterone receptors and leads to disorders of pregnancy. However, in farm animals, such disorders may be one of the reasons leading up to early embryonic lethality, which in cows reaches up to 40%. Obtained results indicate the important role of the studied coregulators in regulating progesterone activity in endometrial cells, especially during the preimplantation period. Therefore, they can be helpful in better understanding the regulation and expression of the coactivators and corepressors in cow endometrium during the estrous cycle and can contribute to reducing this problem. They can also be of significant practical importance, making for the increased efficiency of breeding these animals.Nuclear receptor coregulators include coactivators and corepressors which associate with the progesterone receptor (PGR) during its activation. Fluctuations in the transcription levels of their respective genes and subsequent protein production as well as in related activities for histone acetyltransferase (HAT) and histone deacetylase (HDAC) can affect PGR function and thus change the action of progesterone (P4) in bovine endometrium during the estrous cycle. Endometrial tissue on days 2–5, 6–10, 11–16, and 17–20 of the estrous cycle was used for determination of the mRNA expression levels of coactivators P300, CREB, and SRC-1 along with corepressor NCOR-2 using Real-Time PCR, with protein levels by Western blot. Coregulators cellular localizations were assessed by immunohistochemistry whereas the activities of HAT and HDAC by using EIA. The highest levels of mRNA and proteins for all of the investigated coregulators, as well as the highest levels of activity for HAT and HDAC, were detected over days 2–16 of the estrous cycle. All of the tested coregulatory proteins were localized in the nuclei of endometrial cells. This research indicates the important role of coregulators of the PGR receptor in regulating P4 activity in endometrial cells, especially during the pre-implantation period.