Acetyltransferase Activity Of P300 Research Articles

P300 or CBP are required for calorie restriction-mediated enhancement of skeletal muscle insulin sensitivity

A calorie-restricted diet robustly improves skeletal muscle insulin sensitivity and is a cornerstone lifestyle intervention for preventing and treating clinical hyperglycemia. While studies demonstrate that insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K) and RAC-beta serine/threonine-protein kinase (AKT2) are fundamental to enhanced muscle insulin sensitivity after calorie restriction (CR), the downstream signaling steps remain to be fully elucidated. We have previously demonstrated that the lysine acetyltransferases, E1A binding protein (p300) or cAMP response element binding protein binding protein (CBP) are required for insulin-stimulated glucose uptake by skeletal muscle, at both sub-maximal and maximal concentrations of insulin. Moreover, p300/CBP are phosphorylated by AKT, which increases their acetyltransferase activity. Considering these findings together, the objective of this study was to determine whether p300 or CBP are required for the ability of CR to enhance skeletal muscle insulin sensitivity. We hypothesized that combined inhibition of p300 and CBP acetyltransferase activity would prevent CR-induced enhancement of skeletal muscle insulin sensitivity. To address this objective, 8-week-old male C57BL/6NJ mice were either fed ad libitum (AL; n=16) or were fed a CR diet (60% of AL intake; n=16) diet for 20 days. Then, an ex vivo [3H]-2-deoxyglucose (2DOG) approach was used to assess basal and insulin-stimulated (60μU/mL [0.36nM]) 2DOG uptake in paired extensor digitorum longus (EDL) and soleus from fasted (4 h) mice. To define the role of p300/CBP acetyltransferase activity, paired muscles from AL- or CR-fed mice were pre-incubated for 60 minutes with either DMSO (experimental control; n=8/dietary group [AL-DMSO, CR-DMSO]) or the p300/CBP acetyltransferase activity inhibitor, ip300w (25mM; n=8/dietary group [AL-ip300w, CR-ip300w]). As expected, insulin-stimulated 2DOG uptake (i.e. Insulin 2DOG minus basal 2DOG) in DMSO incubated muscles was ~2.3-fold and ~3.3-fold higher in the soleus and EDL, respectively, from CR- vs. AL-fed mice. Remarkably, ip300w not only blocked insulin-stimulated 2DOG uptake in AL-fed mice, but it also abrogated the insulin-sensitizing effects of CR on muscle insulin sensitivity. To further define the importance of p300 and CBP to CR-induced enhancement of skeletal muscle insulin sensitivity, we studied mice with tamoxifen-inducible and skeletal muscle-specific knockout of CBP and heterozygous (HZ) loss of p300 (referred to as, i-mCKO/PHZ) or knockout of p300 and HZ loss of CBP (referred to as, i-mPKO/CHZ). In these mice, the AL diet or 20 days of CR was initiated 3 weeks after ~10 week-old mice were dosed with tamoxifen (5 consecutive days, 2 mg/day); Cre recombinase-negative littermates, who were also dosed with tamoxifen, were the experimental controls (referred to as wildtype [WT]). Interestingly, oral glucose tolerance and EDL insulin sensitivity were comparably enhanced by CR in WT and both i-mCKO/PHZ and i-mPKO/CHZ mice, thus suggesting that just one allele of p300 or CBP is suffcient for CR to enhance skeletal muscle insulin sensitivity. Taken together, these results demonstrate that p300 or CBP are essential for the ability of calorie restriction to enhance skeletal muscle insulin sensitivity. This work was supported by a grant from the National Institutes of Health (R56 DK1311211), and the Dr. Maryam Ahmadian Memorial Summer Fellowship Program (L.M.L.) and the UC San Diego Medical Scientist Training Program (L.S.F.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anserine, a Histidine-Containing Dipeptide, Suppresses Pressure Overload-Induced Systolic Dysfunction by Inhibiting Histone Acetyltransferase Activity of p300 in Mice.

Anserine, an imidazole dipeptide, is present in the muscles of birds and fish and has various bioactivities, such as anti-inflammatory and anti-fatigue effects. However, the effect of anserine on the development of heart failure remains unknown. We cultured primary cardiomyocytes with 0.03 mM to 10 mM anserine and stimulated them with phenylephrine for 48 h. Anserine significantly suppressed the phenylephrine-induced increases in cardiomyocyte hypertrophy, ANF and BNP mRNA levels, and histone H3K9 acetylation. An in vitro histone acetyltransferase (HAT) assay showed that anserine directly suppressed p300-HAT activity with an IC50 of 1.87 mM. Subsequently, 8-week-old male C57BL/6J mice were subjected to transverse aortic constriction (TAC) and were randomly assigned to receive daily oral treatment with anserine-containing material, Marine Active® (60 or 200 mg/kg anserine) or vehicle for 8 weeks. Echocardiography revealed that anserine 200 mg/kg significantly prevented the TAC-induced increase in left ventricular posterior wall thickness and the decrease in left ventricular fractional shortening. Moreover, anserine significantly suppressed the TAC-induced acetylation of histone H3K9. These results indicate that anserine suppresses TAC-induced systolic dysfunction, at least in part, by inhibiting p300-HAT activity. Anserine may be used as a pharmacological agent for human heart failure therapy.

Near infrared-responsive quinacrine-gold hybrid nanoparticles deregulate HSP-70/P300-mediated H3K14 acetylation in ER/PR+ breast cancer stem cells.

Aim: This study aimed to determine if quinacrine-gold hybrid nanoparticles (QAuNPs)+near-infrared (NIR) deregulate HSP-70/P300 complex-mediated H3K14 acetylation in estrogen receptor/progesterone receptor (ER/PR+) breast cancer stem cells(CSCs). Materials & methods: Various cells and mouse-based systems were used as models. Results: QAuNP+NIR treatment reduced the nuclear translocation of HSP-70, affectedthehistone acetyltransferase activity of P300 and specifically decreased H3K14 acetylation in ER/PR+ breast CSCs. Finally, HSP-70 knockdownshowed a reduction in P300 histone acetyltransferase activity, decreased H3K14 acetylation and inhibited activation of the TGF-β gene. Conclusion: This study revealed that QAuNP+NIR irradiation inhibits oncogenic activation of the TGF-β gene by decreasing H3K14 acetylation mediated through the HSP-70/P300 nuclear complex in ER/PR+ breast CSCs.

In silico screening and identification of potential drug against p300 acetyltransferase activity in breast cancer via drug repurposing approach

Emerging evidence portray the involvement of epigenomic reprogramming in the onset and progression of several malignancies, including breast cancer. Histone acetyltransferase (HAT) p300 is a critical epigenetic regulator that acts as a transcription co-activator and regulates various cellular processes. p300 is overexpressed in breast cancer and promotes cellular invasion and survival, making it a promising druggable target. In this study, the relevance of p300 in different cancer pathways was established. Virtual screening of the FDA-approved drug library was carried out using molecular docking, and the top 10 potential repurposed drugs were identified. Further, recalculation of binding free energy of drug-p300 complexes was carried out using molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) method after molecular dynamic simulation. Based on molecular dynamic simulation parameters and binding free energy analysis, two drugs, namely Netarsudil (−305.068 kJ/mol) and Imatinib (−260.457 kJ/mol), were identified as potential repurposed drugs to inhibit the activity of p300. In conclusion, these findings suggest, Netarsudil and Imatinib might be a potential repurposed drug to combat breast cancer via p300 inhibition. Communicated by Ramaswamy H. Sarma

Regulation of HDAC11 gene expression in early myogenic differentiation.

Histone acetylation and deacetylation affect the patterns of gene expression in cellular differentiation, playing pivotal roles in tissue development and maintenance. For example, the intrinsic histone acetyltransferase activity of transcriptional coactivator p300 is especially required for the expression of myogenic regulatory factors including Myf5 and MyoD, and consequently for skeletal myogenesis. On the other hand, histone deacetylases (HDACs) remove the acetyl group from histones, which is critical for gene repression in stem cell fate transition. Through integrative omic analyses, we found that while some HDACs were differentially expressed at the early stage of skeletal myoblast differentiation, Hdac11 gene expression was significantly enhanced by nuclear receptor signaling. In addition, p300 and MyoD control Hdac11 expression in milieu of normal and signal-enhanced myoblast differentiation. Thus, HDAC11 may be essential to differential gene expression at the onset of myoblast differentiation.

PS-BPB02-8: CURCUMIN, AN INHIBITOR OF P300-HAT ACTIVITY, PREVENTS HYPERTENSION-INDUCED CARDIAC HYPERTROPHY WITH PRESERVED SYSTOLIC FUNCTION IN DAHL RATS

Background: Hypertension causes left ventricular hypertrophy (LVH) with a preserved systolic function. While LVH might be a compensatory process, its presence of LVH is associated with an increased incidence of cardiovascular events and heart failure. Recently, we found that curcumin, a major curcuminoid isolated from Curcuma longa, inhibits the histone acetyltransferase (HAT) activity of p300 as well as the p300-induced acetylation of histones and a hypertrophy-responsive transcription factor, GATA-4, in cultured cardiomyocytes. Furthermore, curcumin prevents deterioration of the systolic function in rat heart failure models in vivo. However, whether curcumin prevents the development of hypertension-induced LVH at an early stage of hypertension is unclear. Objective: To solve this problem, we have utilized a salt-sensitive Dahl rat (DS) model of hypertension. Methods: We randomized 6-week-old DS (n = 19) and control salt-resistant Dahl rats (DR, n = 10) to curcumin or the vehicle group. Then, these rats were given a high-salt diet and subjected to daily oral treatment with 50 mg/kg/day of curcumin or its vehicle (1% Gum arabic) for 6 weeks. Results: There were no differences between curcumin and vehicle in any data examined before treatment (6 weeks of age). The high-salt diet-induced a similar degree of hypertension in these two groups of DS compared with DR. At 12 weeks of age, transthoracic echocardiography in the vehicle group showed that the LV wall thickness, LV mass, and LV fractional shortening were significantly higher in DS compared with DR, and the LV systolic and diastolic dimensions were significantly smaller in DS than DR. Curcumin treatment significantly (p < 0.01) decreased the LV wall thickness in DS but not DR. Curcumin also significantly (p < 0.01) decreased the LV mass in DS, but not in DR. However, LV fractional shortening in DS was similar between the two groups. Curcumin also prevented the increases in hypertension-induced myocardial cell hypertrophy and perivascular fibrosis. Furthermore, curcumin significantly attenuated the up-regulation of hypertrophy-response gene transcriptions as well as acetylation of GATA4 but not phosphorylation of ERK1/2 and GATA4 in the heart. Conclusions: The natural compound, curcumin, inhibits the development of hypertension-induced concentric LVH without affecting the systolic function through inhibition of GATA4-acetylation. Thus, this compound might be applicable to patients with early-stages hypertensive heart diseases.

Abstract 10318: Polyunsaturated Fatty Acids, EPA and DHA Possess a Potent P300-hat Inhibitor and Prevent Mi-Induced Development of Heart Failure in Rats

Introduction: The histone acetyltransferase (HAT) activity of p300 has a crucial role in the development of heart failure. Although many studies have shown a cardioprotective effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), omega-3 unsaturated fatty acids, little is known about the effects of these acids on cardiomyocyte hypertrophy. In this study, we investigated the effects of these acids on cardiomyocyte hypertrophy and the development of heart failure in rats with myocardial infarction (MI). Methods and Results: In cardiomyocytes, EPA and DHA (10 μM) significantly suppressed equally PE-induced cardiomyocyte hypertrophy, hypertrophy-response gene transcriptions, and acetylation of histone-H3K9. Furthermore, p300-induced hypertrophic responses were also suppressed by EPA and DHA. The result of an in vitro p300-HAT assay revealed that EPA and DHA directly inhibited p300-HAT activity (IC50: 37.8 μM, 30.6 μM) and induced allosteric inhibition of histones and competitive inhibition of acetyl-CoA in vitro . In addition, palmitic acid and stearic acid could not inhibit them. MI-operated rats (FS<40%) were randomly assigned to 3 groups; vehicle (saline), EPA, and DHA (1g/kg). One week after the operation, oral administrations were repeated for 6 weeks. The echocardiographic analysis demonstrated that both EPA and DHA treatments prevented MI-induced left ventricular remodeling with preserved FS. Furthermore, EPA and DHA significantly suppressed the MI-induced increase in myocardial cell diameter, perivascular fibrosis, mRNA levels of hypertrophic and fibrotic markers, and acetylation of histone H3K9. In the left ventricle, EPA contents were lower than those of the other fatty acids, and no difference in each group. The EPA group exhibited higher docosapentaenoic acid (DPA) and DHA content than the vehicle group. The DHA group increased DHA content but not DPA and EPA contents. Conclusion: These results suggested that both EPA and DHA suppressed MI-induced development of heart failure through the inhibition of p300-HAT activity. The cardioprotective effect of EPA on MI might be attributed to the conversion of DHA or EPA metabolites.

Methods to Evaluate the Effects of HAT/KAT Inhibition on SIAH2-Driven Reactive Oxygen Species Generation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori infection is one of the leading factors that promotes, among other diseases, gastric cancer (GC). Infection of gastric epithelial cells (GECs) by H. pylori enhances the expression as well as acetylation of the E3 ubiquitin ligase SIAH2 which promotes GC progression. The histone acetyltransferase (HAT) activity of p300 catalyzes SIAH2 acetylation following H. pylori infection. Since reactive oxygen species (ROS) generation in H. pylori-infected GECs accelerates GC progression, acetylation-mediated SIAH2 regulation might be a crucial modifier of ROS generation in the infected GECs. Here, we describe a compendium of methods to evaluate the effects of HAT/lysine acetyl transferase (KAT) inhibitors (HAT/KATi) on SIAH2-mediated ROS regulation in H. pylori-infected GECs.

SIK2 protects against renal tubular injury and the progression of diabetic kidney disease

Despite optimal medical therapy, many patients with diabetic kidney disease (DKD) progress to end-stage renal disease. The identification of new biomarkers and drug targets for DKD is required for the development of more effective therapies. The apoptosis of renal tubular epithelial cells is a key feature of the pathogenicity associated with DKD. SIK2, a salt-inducible kinase, regulates important biological processes, such as energy metabolism, cell cycle progression and cellular apoptosis. In our current study, a notable decrease in the expression of SIK2 was detected in the renal tubules of DKD patients and murine models. Functional experiments demonstrated that deficiency or inactivity of SIK2 aggravates tubular injury and interstitial fibrosis in diabetic mice. Based on transcriptome sequencing, molecular mechanism exploration revealed that SIK2 overexpression reduces endoplasmic reticulum (ER) stress-mediated tubular epithelial apoptosis by inhibiting the histone acetyltransferase activity of p300 to activate HSF1/Hsp70. Furthermore, the specific restoration of SIK2 in tubules blunts tubular and interstitial impairments in diabetic and vancomycin-induced kidney disease mice. Together, these findings indicate that SIK2 protects against renal tubular injury and the progression of kidney disease, and make a compelling case for targeting SIK2 for therapy in DKD.

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.

P300 suppresses the transition of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are hematopoietic stem and progenitor cell (HSPC) malignancies characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Epigenetic regulators are recurrently mutated in MDS, directly implicating epigenetic dysregulation in MDS pathogenesis. Here, we identified a tumor suppressor role of the acetyltransferase p300 in clinically relevant MDS models driven by mutations in the epigenetic regulators TET2, ASXL1, and SRSF2. The loss of p300 enhanced the proliferation and self-renewal capacity of Tet2-deficient HSPCs, resulting in an increased HSPC pool and leukemogenicity in primary and transplantation mouse models. Mechanistically, the loss of p300 in Tet2-deficient HSPCs altered enhancer accessibility and the expression of genes associated with differentiation, proliferation, and leukemia development. Particularly, p300 loss led to an increased expression of Myb, and the depletion of Myb attenuated the proliferation of HSPCs and improved the survival of leukemia-bearing mice. Additionally, we show that chemical inhibition of p300 acetyltransferase activity phenocopied Ep300 deletion in Tet2-deficient HSPCs, whereas activation of p300 activity with a small molecule impaired the self-renewal and leukemogenicity of Tet2-deficient cells. This suggests a potential therapeutic application of p300 activators in the treatment of MDS with TET2 inactivating mutations.

Nuclear ADP-ribosylation drives IFN\u03b3-dependent STAT1\u03b1 enhancer formation in macrophages

STAT1α is a key transcription factor driving pro-inflammatory responses in macrophages. We found that the interferon gamma (IFNγ)-regulated transcriptional program in macrophages is controlled by ADP-ribosylation (ADPRylation) of STAT1α, a post-translational modification resulting in the site-specific covalent attachment of ADP-ribose moieties. PARP-1, the major nuclear poly(ADP-ribose) polymerase (PARP), supports IFNγ-stimulated enhancer formation by regulating the genome-wide binding and IFNγ-dependent transcriptional activation of STAT1α. It does so by ADPRylating STAT1α on specific residues in its DNA-binding domain (DBD) and transcription activation (TA) domain. ADPRylation of the DBD controls STAT1α binding to its cognate DNA elements, whereas ADPRylation of the TA domain regulates enhancer activation by modulating STAT1α phosphorylation and p300 acetyltransferase activity. Loss of ADPRylation at either site leads to diminished IFNγ-dependent transcription and downstream pro-inflammatory responses. We conclude that PARP-1-mediated ADPRylation of STAT1α drives distinct enhancer activation mechanisms and is a critical regulator of inflammatory responses in macrophages.

Carnosol Is a Novel Inhibitor of p300 Acetyltransferase in Breast Cancer.

Carnosol, a natural polyphenol abundant in edible plants such as sage, rosemary, and oregano, has shown promising anticancer activity against various types of cancers. Nonetheless, very little is known about its molecular mechanism of action or its downstream target(s). We have previously shown that carnosol inhibits cellular proliferation, migration, invasion, and metastasis as well as triggers autophagy and apoptosis in the highly invasive MDA-MB-231 breast cancer cells. Here, we report that carnosol induces histone hypoacetylation in MDA-MB-231 and Hs578T breast cancer cells. We show that, while carnosol does not affect HDACs, it promotes a ROS-dependent proteasome degradation of p300 and PCAF histone acetyl transferases (HATs) without affecting other HATs such as GCN5 and hMOF. Carnosol-induced histone hypoacetylation remains persistent even when p300 and PCAF protein levels were rescued from degradation by (i) the inhibition of the proteasome activity by the proteasome inhibitors MG-132 and bortezomib, and (ii) the inhibition of ROS accumulation by the ROS scavenger, N-acetylcysteine. In addition, we report that, in a cell-free system, carnosol efficiently inhibits histone acetyltransferase activity of recombinant p300 but not that of PCAF or GCN5. Molecular docking studies reveal that carnosol inhibits p300 HAT activity by blocking the entry of the acetyl-CoA binding pocket of the catalytic domain. The superimposition of the docked conformation of the p300 HAT domain in complex with carnosol shows a similar orientation as the p300 structure with acetyl-CoA. Carnosol occupies the region where the pantetheine arm of the acetyl-CoA is bound. This study further confirms carnosol as a promising anti-breast cancer therapeutic compound and identifies it as a novel natural p300 inhibitor that could be added to the existing panel of inhibitors.

Alterations of Gut Microbiota by Overnutrition Impact Gluconeogenic Gene Expression and Insulin Signaling.

A high-fat, Western-style diet is an important predisposing factor for the onset of type 2 diabetes and obesity. It causes changes in gut microbial profile, reduction of microbial diversity, and the impairment of the intestinal barrier, leading to increased serum lipopolysaccharide (endotoxin) levels. Elevated lipopolysaccharide (LPS) induces acetyltransferase P300 both in the nucleus and cytoplasm of liver hepatocytes through the activation of the IRE1-XBP1 pathway in the endoplasmic reticulum stress. In the nucleus, induced P300 acetylates CRTC2 to increase CRTC2 abundance and drives Foxo1 gene expression, resulting in increased expression of the rate-limiting gluconeogenic gene G6pc and Pck1 and abnormal liver glucose production. Furthermore, abnormal cytoplasm-appearing P300 acetylates IRS1 and IRS2 to disrupt insulin signaling, leading to the prevention of nuclear exclusion and degradation of FOXO1 proteins to further exacerbate the expression of G6pc and Pck1 genes and liver glucose production. Inhibition of P300 acetyltransferase activity by chemical inhibitors improved insulin signaling and alleviated hyperglycemia in obese mice. Thus, P300 acetyltransferase activity appears to be a therapeutic target for the treatment of type 2 diabetes and obesity.

SL010110, a lead compound, inhibits gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and improves glucose homeostasis in diabetic mice.

Suppression of excessive hepatic gluconeogenesis is an effective strategy for controlling hyperglycemia in type 2 diabetes (T2D). In the present study, we screened our compounds library to discover the active molecules inhibiting gluconeogenesis in primary mouse hepatocytes. We found that SL010110 (5-((4-allyl-2-methoxyphenoxy) methyl) furan-2-carboxylic acid) potently inhibited gluconeogenesis with 3 μM and 10 μM leading to a reduction of 45.5% and 67.5%, respectively. Moreover, SL010110 caused suppression of gluconeogenesis resulted from downregulating the protein level of phosphoenolpyruvate carboxykinase 1 (PEPCK1), but not from affecting the gene expressions of PEPCK, glucose-6-phosphatase, and fructose-1,6-bisphosphatase. Furthermore, SL010110 increased PEPCK1 acetylation, and promoted PEPCK1 ubiquitination and degradation. SL010110 activated p300 acetyltransferase activity in primary mouse hepatocytes. The enhanced PEPCK1 acetylation and suppressed gluconeogenesis caused by SL010110 were blocked by C646, a histone acetyltransferase p300 inhibitor, suggested that SL010110 inhibited gluconeogenesis by activating p300. SL010110 decreased NAD+/NADH ratio, inhibited SIRT2 activity, and further promoted p300 acetyltransferase activation and PEPCK1 acetylation. These effects were blocked by NMN, an NAD+ precursor, suggested that SL010110 inhibited gluconeogenesis by inhibiting SIRT2, activating p300, and subsequently promoting PEPCK1 acetylation. In type 2 diabetic ob/ob mice, single oral dose of SL010110 (100 mg/kg) suppressed gluconeogenesis accompanied by the suppressed hepatic SIRT2 activity, increased p300 activity, enhanced PEPCK1 acetylation and degradation. Chronic oral administration of SL010110 (15 or 50 mg/kg) significantly reduced the blood glucose levels in ob/ob and db/db mice. This study reveals that SL010110 is a lead compound with a distinct mechanism of suppressing gluconeogenesis via SIRT2-p300-mediated PEPCK1 degradation and potent anti-hyperglycemic activity for the treatment of T2D.

SIRT2 Inhibition Improves Functional Motor Recovery After Peripheral Nerve Injury.

Sirtuin-2 (Sirt2) is a member of the NAD (+)-dependent protein deacetylase family involved in neuroprotection, cellular metabolism, homeostasis, and stress responses after injury of the nervous system. So far, no data have been published describing the role of SIRT2 in motor functional recovery after damage. We found that SIRT2 expression and deacetylase activity were increased within motoneurons after axotomy. To shed light onto the biological relevance of this change, we combined in vitro and in vivo models with pharmacological and genetic ablation approaches. We found that SIRT2 KO (knockout) mice exhibited improved functional recovery after sciatic nerve crush. SIRT2 activity blockage, using AK7, increased neurite outgrowth and length in organotypic spinal cord cultures and human cell line models. SIRT2 blockage enhanced the acetyltransferase activity of p300, which in turn increased the levels of an acetylated form of p53 (Ac-p53 k373), histone 3 (Ac-H3K9), and expression of GAP43, a downstream marker of regeneration. Lastly, we verified that p300 acetyltransferase activity is essential for these effects. Our results suggest that bolstering an epigenetic shift that promotes SIRT2 inhibition can be an effective therapy to increase functional recovery after peripheral nerve injury.

The acetyltransferase p300 regulates NRF2 stability and localization

The transcription factor NRF2 plays a key role in the protection against environmental stress and maintaining cellular homeostasis. The acetyltransferase p300 is a known component of the NRF2 transcriptional complex and promotes its transcriptional activity. In this study we describe a novel mechanism by which p300 facilitates NRF2 activity. p300 physically interacts with NRF2 and interferes with NRF2-KEAP1 complex formation. In particular, p300 increases NRF2 protein abundance and stability, thereby promoting NRF2 nuclear localization. Notably, the acetyltransferase activity of p300 was indispensable for the stabilizing effects towards NRF2. Furthermore, overexpression of p300 protected HEK293T cells from oxidative stress and increased viability. Together our study uncovers a link between p300 and control of NRF2-KEAP1 signaling via regulation of NRF2 stability and this may act as a novel checkpoint on the adaptation to oxidative stress.

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

P5434P300-mediated inactivation of p53 protects against doxorubicin-induced cardiotoxicity

Abstract Introduction Doxorubicin is amongst the most widely prescribed chemotherapy drugs due to its effectiveness in cancer treatment. However, progressive treatment using doxorubicin severely increases the risk of congestive heart failure. Mechanistically, researchers have shown that doxorubicin (i) intercalates into DNA and disrupts topoisomerase-II-mediated DNA repair and (ii) generates free radicals which then causes damage to cellular membranes, DNA, and proteins. Ultimately these cellular insults induce cardiomyocyte (CM) death mediated by the tumour suppressor, p53. Currently, there are no clinically applicable preventative treatments for doxorubicin-induced cardiotoxicity and so, extensive research is being done in discovering a potential therapy. One such candidate is curcumin – a natural polyphenol compound non-toxic to humans. We have previously demonstrated that curcumin inhibits lysine acetyltransferase activity of p300, therefore reducing both histone and non-histone protein acetylation. To induce CM death, p53 requires acetylation by p300. Therefore, we hypothesize that curcumin protects against doxorubicin-induced CM death and cardiotoxicity via p300-mediated inactivation of p53. Methods Rat H9c2 cardiomyoblast cells were cultured and treated with a 2.5 μM dose of doxorubicin for 16 hours. One group of cells were pre-treated with curcumin (15μM) 4 hours prior to doxorubicin treatment, and controls were cultured with only diluent added. Following treatment, the cells were harvested for total protein. At end point, we performed immunoblotting to measure protein expression of key proteins involved in DNA damage (y-H2A.X, p53), and apoptosis (cleaved-Caspase 3). Results Our findings show that following doxorubicin treatment, p53 expression was significantly increased (p<0.001), confirming its role in doxorubicin-associated cardiotoxicity. Furthermore, doxorubicin was associated with increased DNA-damage as evidenced by increased expression and activation of DNA double-stranded breaks (DSBs) marker, y-H2A.X (p<0.001). Elevated levels of DNA-damage were further associated with significantly increased doxorubicin-induced apoptosis as measured by immunoblotting for cleaved-Caspase 3 (p<0.001). However, DNA-damage and apoptosis were attenuated by pre-treatment of cells with curcumin. Curcumin treatment led to a significant decrease in both y-H2A.X (p<0.01) and cleaved-Caspase 3 (p<0.01) expression compared to cells treated with only doxorubicin. Conclusions Our data provides the first evidence that curcumin protects against doxorubicin-induced apoptosis in rat cardiomyoblast cells in vitro. Further studies are warranted in vivo to confirm the potential of curcumin as a cardio-protective drug. Curcumin is a natural compound with little to no side-effects in humans, therefore our finding may provide a novel therapeutic target and treatment approach for doxorubicin-associated cardiotoxicity. Acknowledgement/Funding Heart & Stroke Foundation of Canada

P300 Acetylates JHDM1A to inhibit osteosarcoma carcinogenesis

JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.