Deacetylase Activity Research Articles

Ameliorative inhibition of sirtuin 6 by imidazole derivative triggers oxidative stress-mediated apoptosis associated with Nrf2/Keap1 signaling in non-small cell lung cancer cell lines.

Background: Redox homeostasis is the vital regulatory system with respect to antioxidative response and detoxification. The imbalance of redox homeostasis causes oxidative stress. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, also called Nfe2l2)/Kelchlike ECH-associated protein 1 (Keap1) signaling is the major regulator of redox homeostasis. Nrf2/Keap1 signaling is reported to be involved in cancer cell growth and survival. A high level of Nrf2 in cancers is associated with poor prognosis, resistance to therapeutics, and rapid proliferation, framing Nrf2 as an interesting target in cancer biology. Sirtuins (SIRT1-7) are class III histone deacetylases with NAD + dependent deacetylase activity that have a remarkable impact on antioxidant and redox signaling (ARS) linked with Nrf2 deacetylation thereby increasing its transcription by epigenetic modifications which has been identified as a crucial event in cancer progression under the influence of oxidative stress in various transformed cells. SIRT6 plays an important role in the cytoprotective effect of multiple diseases, including cancer. This study aimed to inhibit SIRT6 using an imidazole derivative, Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate, to assess its impact on Nrf2/Keap1 signaling in A549 and NCI-H460 cell lines. Method: Half maximal inhibitory concentration (IC50) of Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate was fixed by cell viability assay. The changes in the gene expression of important regulators involved in this study were examined using quantitative real-time PCR (qRT-PCR) and protein expression changes were confirmed by Western blotting. The changes in the antioxidant molecules are determined by biochemical assays. Further, morphological studies were performed to observe the generation of reactive oxygen species, mitochondrial damage, and apoptosis. Results: We inhibited SIRT6 using Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate and demonstrated that SIRT6 inhibition impacts the modulation of antioxidant and redox signaling. The level of antioxidant enzymes and percentage of reactive oxygen species scavenging activity were depleted. The morphological studies showed ROS generation, mitochondrial damage, nuclear damage, and apoptosis. The molecular examination of apoptotic factors confirmed apoptotic cell death. Further, molecular studies confirmed the changes in Nrf2 and Keap1 expression during SIRT6 inhibition. Conclusion: The overall study suggests that SIRT6 inhibition by imidazole derivative disrupts Nrf2/Keap1 signaling leading to oxidative stress and apoptosis induction.

LncRNA DANA1 promotes drought tolerance and histone deacetylation of drought responsive genes in Arabidopsis

Although many long noncoding RNAs have been discovered in plants, little is known about their biological function and mode of action. Here we show that the drought-induced long intergenic noncoding RNA DANA1 interacts with the L1p/L10e family member protein DANA1-INTERACTING PROTEIN 1 (DIP1) in the cell nucleus of Arabidopsis, and both DANA1 and DIP1 promote plant drought resistance. DANA1 and DIP1 increase histone deacetylase HDA9 binding to the CYP707A1 and CYP707A2 loci. DIP1 further interacts with PWWP3, a member of the PEAT complex that associates with HDA9 and has histone deacetylase activity. Mutation of DANA1 enhances CYP707A1 and CYP707A2 acetylation and expression resulting in impaired drought tolerance, in agreement with dip1 and pwwp3 mutant phenotypes. Our results demonstrate that DANA1 is a positive regulator of drought response and that DANA1 works jointly with the novel chromatin-related factor DIP1 on epigenetic reprogramming of the plant transcriptome during the response to drought.

Butyric acid and prospects for creation of new medicines based on its derivatives: a literature review.

The widespread use of antimicrobials causes antibiotic resistance in bacteria. The use of butyric acid and its derivatives is an alternative tactic. This review summarizes the literature on the role of butyric acid in the body and provides further prospects for the clinical use of its derivatives and delivery methods to the animal body. Thus far, there is evidence confirming the vital role of butyric acid in the body and the effectiveness of its derivatives when used as animal medicines and growth stimulants. Butyric acid salts stimulate immunomodulatory activity by reducing microbial colonization of the intestine and suppressing inflammation. Extraintestinal effects occur against the background of hemoglobinopathy, hypercholesterolemia, insulin resistance, and cerebral ischemia. Butyric acid derivatives inhibit histone deacetylase. Aberrant histone deacetylase activity is associated with the development of certain types of cancer in humans. Feed additives containing butyric acid salts or tributyrin are used widely in animal husbandry. They improve the functional status of the intestine and accelerate animal growth and development. On the other hand, high concentrations of butyric acid stimulate the apoptosis of epithelial cells and disrupt the intestinal barrier function. This review highlights the biological activity and the mechanism of action of butyric acid, its salts, and esters, revealing their role in the treatment of various animal and human diseases. This paper also discussed the possibility of using butyric acid and its derivatives as surface modifiers of enterosorbents to obtain new drugs with bifunctional action.

Isthmin-1 Improves Aging-Related Cardiac Dysfunction in Mice through Enhancing Glycolysis and SIRT1 Deacetylase Activity.

Aging-related cardiac dysfunction poses a major risk factor of mortality for elderly populations, however, efficient treatment for aging-related cardiac dysfunction is far from being known. Isthmin-1 (ISM1) is a novel adipokine that promotes glucose uptake and acts indispensable roles in restraining inflammatory and fibrosis. The present study aims to investigate the potential role and molecular mechanism of ISM1 in aging-related cardiac dysfunction. Aged and matched young mice were overexpressed or silenced with ISM1 to investigate the role of ISM1 in aging-related cardiac dysfunction. Moreover, H9C2 cells were stimulated with D-galactose (D-gal) to examine the role of ISM1 in vitro. Herein, we found that cardiac-specific overexpression of ISM1 significantly mitigated insulin resistance by promoting glucose uptake in aging mice. ISM1 overexpression alleviated while ISM1 silencing deteriorated cellular senescence, cardiac inflammation, and dysfunction in natural and accelerated cardiac aging. Mechanistically, ISM1 promoted glycolysis and activated Sirtuin-1 (SIRT1) through increasing glucose uptake. ISM1 increased glucose uptake via translocating GLUT4 to the surface, thereby enhancing glycolytic flux and hexosamine biosynthetic pathway (HBP) flux, ultimately leading to increased SIRT1 activity through O-GlcNAc modification. ISM1 may serve as a novel potential therapeutic target for preventing aging-related cardiac disease in elderly populations. ISM1 prevents aging-related cardiac dysfunction by promoting glycolysis and enhancing SIRT1 deacetylase activity, making it a promising therapeutic target for aging-related cardiac disease.

Emerging role of HDAC11 in skeletal muscle biology.

HDAC11 is an epigenetic repressor of gene transcription, acting through its deacetylase activity to remove functional acetyl groups from the lysine residues of histones at genomic loci. It has been implicated in the regulation of different immune responses, metabolic activities, as well as cell cycle progression. Recent studies have also shed lights on the impact of HDAC11 on myogenic differentiation and muscle development, indicating that HDAC11 is important for histone deacetylation at the promoters to inhibit transcription of cell cycle related genes, thereby permitting myogenic activation at the onset of myoblast differentiation. Interestingly, the upstream networks of HDAC11 target genes are mainly associated with cell cycle regulators and the acetylation of histones at the HDAC11 target promoters appears to be residue specific. As such, selective inhibition, or activation of HDAC11 presents a potential therapeutic approach for targeting distinct epigenetic pathways in clinical applications.

Histone Trimethylations and HDAC5 Regulate Spheroid Subpopulation and Differentiation Signaling of Human Adipose-Derived Stem Cells.

Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation.

Mechanism by which HDAC3 regulates manganese induced H3K27ac in SH-SY5Y cells and intervention by curcumin

Long-term excessive exposure to manganese can impair neuronal function in the brain, but the underlying pathological mechanism remains unclear. Oxidative stress plays a central role in manganese-induced neurotoxicity. Numerous studies have established a strong link between abnormal histone acetylation levels and the onset of various diseases. Histone deacetylase inhibitors and activators, such as TSA and ITSA-1, are often used to investigate the intricate mechanisms of histone acetylation in disease. In addition, recent experiments have provided substantial evidence demonstrating that curcumin (Cur) can act as an epigenetic regulator. Given these findings, this study aims to investigate the mechanisms underlying oxidative damage in SH-SY5Y cells exposed to MnCl2·4H2O, with a particular focus on histone acetylation, and to assess the potential therapeutic efficacy of Cur. In this study, SH-SY5Y cells were exposed to manganese for 24 h, were treated with TSA or ITSA-1, and were treated with or without Cur. The results suggested that manganese exposure, which leads to increased expression of HDAC3, induced H3K27 hypoacetylation, inhibited the transcription of antioxidant genes, decreased antioxidant enzyme activities, and induced oxidative damage in cells. Pretreatment with an HDAC3 inhibitor (TSA) increased the acetylation of H3K27 and the transcription of antioxidant genes and thus slowed manganese exposure-induced cellular oxidative damage. In contrast, an HDAC3 activator (ITSA-1) partially increased manganese-induced cellular oxidative damage, while Cur prevented manganese-induced oxidative damage. In summary, these findings suggest that inhibiting H3K27ac is a possible mechanism for ameliorating manganese-induced damage to dopaminergic neurons and that Cur exerts a certain protective effect against manganese-induced damage to dopaminergic neurons.

Structural Insights into the Catalytic Activity of Cyclobacterium marinum N-Acetylglucosamine Deacetylase.

N-Acetylglucosamine deacetylase from Cyclobacterium marinum (CmCBDA) is a highly effective and selective biocatalyst for the production of d-glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). However, the underlying catalytic mechanism remains elusive. Here, we show that CmCBDA is a metalloenzyme with a preference for Ni2+ over Mn2+. Crystal structures of CmCBDA in complex with Ni2+ and Mn2+ revealed slight remodeling of the CmCBDA active site by the metal ions. We also demonstrate that CmCBDA exists as a mixture of homodimers and monomers in solution, and dimerization is indispensable for catalytic activity. A mutagenesis analysis also indicated that the active site residues Asp22, His72, and His143 as well as the residues involved in dimerization, Pro52, Trp53, and Tyr55, are essential for catalytic activity. Furthermore, a mutation on the protein surface, Lys219Glu, resulted in a 2.3-fold improvement in the deacetylation activity toward GlcNAc. Mechanistic insights obtained here may facilitate the development of CmCBDA variants with higher activities.

ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD), which still lacks of reliable markers and therapeutic options. Cellular senescence has been considered an important mechanism of intestinal fibrosis, but the underlying molecular link remains elusive. Tissues were stained using α-smooth muscle actin (α-SMA), fibronectin, and collagen I as markers of myofibroblastic differentiation. Cellular senescence was confirmed through Lamin B1 staining, senescence-associated β-galactosidase staining, and the expression of senescence-associated secretory phenotype (SASP) factors. We explored the relationship between senescence of intestinal epithelial cells (IECs) and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. The effects of irisin on cellular senescence and fibrosis were determined. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a novel biomarker for intestinal cellular senescence and fibrosis. In fibrostrictured tissues from patients and murine models with IBD, significantly high levels of cellular senescence score and factors were noted, which positively correlated with the fibrotic regulator fibronectin. Senescent IECs, not fibroblast itself, released SASP factors to regulate fibroblast activation. Prolonging exposure to severe and persistent injurious stimuli decreased ELMO1 expression, which dampened SIRT1 deacetylase activity, enhanced NF-κB (p65) acetylation, and thereby accelerated cellular senescence. Deletion of ELMO1 led to senescent IECs accumulation and triggered premature fibrosis in murine colitis. Furthermore, irisin, inhibiting the degradation of ELMO1, could downregulate p65 acetylation, reduce IECs senescence, and prevent incipient intestinal fibrosis in murine colitis models. This study reveals ELMO1 downregulation is an early symbol of intestinal senescence and fibrosis, and the altered ELMO1-SIRT1-p65 pathway plays an important role in intestinal cellular senescence and IBD-related fibrosis.

LONG-TERM NMN TREATMENT INCREASES LIFESPAN AND HEALTHSPAN IN MICE IN A SEX DEPENDENT MANNER

Abstract Levels of nicotinamide adenine dinucleotide (NAD+) decline by up to 50% in aging. NAD+ is an essential co-factor for many metabolic processes, including the deacetylase activity of sirtuins, and previous studies have demonstrated that supplementing NAD+ levels has a range of health benefits in both mice and humans. Here we investigate the effect of long-term (from 13 months of age) administration of the NAD+ precursor nicotinamide mononucleotide (NMN) on frailty and lifespan in male and female mice. NMN treatment delayed the onset of frailty in both sexes, improved metabolic health in male mice, and increased median lifespan by 8.5% in female mice. Exploration of the potential mechanisms of this protection showed that NMN treatment prevented age-related gene expression changes in skeletal muscle and led to a large increase in levels of Anaerotruncus colihominis, a microbe associated with reduced inflammation, in the gut. A thorough characterization of NMN metabolism across age, sex and tissues shows context-specific sex differences in metabolic pathways, including greater Preiss-Handler pathway metabolism in females than males, which may contribute to observed sex differences in health and lifespan. Overall, this data provides preclinical evidence that chronic NMN treatment increases lifespan and improves frailty and metabolic health in aging, and highlights the importance of using both sexes for interventional lifespan studies.

A Sirtuin-Dependent T7 RNA Polymerase Variant.

Transcriptional regulation is of great significance for cells to maintain homeostasis and, meanwhile, represents an innovative but less explored means to control biological processes in synthetic biology and bioengineering. Herein we devised a T7 RNA polymerase (T7RNAP) variant through replacing an essential lysine located in the catalytic core (K631) with Nε-acetyl-l-lysine (AcK) via genetic code expansion. This T7RNAP variant requires the deacetylase activity of NAD-dependent sirtuins to recover its enzymatic activities and thereby sustains sirtuin-dependent transcription of the gene of interest in live cells including bacteria and mammalian cells as well as in in vitro systems. This T7RNAP variant could link gene transcription to sirtuin expression and NAD availability, thus holding promise to support some relevant research.

Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

BackgroundMetabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most prevalent metabolic syndromes worldwide. However, no approved pharmacological treatments are available for MAFLD. Chenpi, one kind of dried peel of citrus fruits, has traditionally been utilized as a medicinal herb for liver diseases. Didymin is a newly identified oral bioactive dietary flavonoid glycoside derived from Chenpi. In this study, we investigated the therapeutic potential of Didymin as an anti-MAFLD drug and elucidated its underlying mechanisms.MethodsHigh-fat diet (HFD)-induced MAFLD mice and alpha mouse liver 12 (AML12) cells were utilized to evaluate the effects and mechanisms of Didymin in the treatment of MAFLD. Liver weight, serum biochemical parameters, and liver morphology were examined to demonstrate the therapeutic efficacy of Didymin in MAFLD treatment. RNA-seq analysis was performed to identify potential pathways that could be affected by Didymin. The impact of Didymin on Sirt1 was corroborated through western blot, molecular docking analysis, microscale thermophoresis (MST), and deacetylase activity assay. Then, a Sirt1 inhibitor (EX-527) was utilized to confirm that Didymin alleviates MAFLD via Sirt1. Western blot and additional assays were used to investigate the underlying mechanisms.ResultsOur results suggested that Didymin may possess therapeutic potential against MAFLD in vitro and in vivo. By promoting Sirt1 expression as well as directly binding to and activating Sirt1, Didymin triggers downstream pathways that enhance mitochondrial biogenesis and function while reducing apoptosis and enhancing lipophagy.ConclusionsThese suggest that Didymin could be a promising medication for MAFLD treatment. Furthermore, its therapeutic effects are mediated by Sirt1.

A distinct isoform of lymphoid enhancer binding factor 1 (LEF1) epigenetically restricts EBV reactivation to maintain viral latency.

As a human tumor virus, EBV is present as a latent infection in its associated malignancies where genetic and epigenetic changes have been shown to impede cellular differentiation and viral reactivation. We reported previously that levels of the Wnt signaling effector, lymphoid enhancer binding factor 1 (LEF1) increased following EBV epithelial infection and an epigenetic reprogramming event was maintained even after loss of the viral genome. Elevated LEF1 levels are also observed in nasopharyngeal carcinoma and Burkitt lymphoma. To determine the role played by LEF1 in the EBV life cycle, we used in silico analysis of EBV type 1 and 2 genomes to identify over 20 Wnt-response elements, which suggests that LEF1 may bind directly to the EBV genome and regulate the viral life cycle. Using CUT&RUN-seq, LEF1 was shown to bind the latent EBV genome at various sites encoding viral lytic products that included the immediate early transactivator BZLF1 and viral primase BSLF1 genes. The LEF1 gene encodes various long and short protein isoforms. siRNA depletion of specific LEF1 isoforms revealed that the alternative-promoter derived isoform with an N-terminal truncation (ΔN LEF1) transcriptionally repressed lytic genes associated with LEF1 binding. In addition, forced expression of the ΔN LEF1 isoform antagonized EBV reactivation. As LEF1 repression requires histone deacetylase activity through either recruitment of or direct intrinsic histone deacetylase activity, siRNA depletion of LEF1 resulted in increased histone 3 lysine 9 and lysine 27 acetylation at LEF1 binding sites and across the EBV genome. Taken together, these results indicate a novel role for LEF1 in maintaining EBV latency and restriction viral reactivation via repressive chromatin remodeling of critical lytic cycle factors.

Blockade of histone deacetylase activity affects transcription and splicing of neuronal and glial genes

The study of the molecular mechanisms underlying plastic processes in the nervous system is of great interest in modern neuroscience. It is important to understand that epigenetic modifications, which are crucial for the development and cellular differentiation, can also be involved in plastic processes in the adult nervous system. In our early work, we provided evidence that the expression of important memory-related genes, such as Prkcz and Prkci, can be regulated epigenetically [1]. In the current study, we extended the previous work to the systemic level by applying the RNA sequencing approach to evaluate global changes in the expression patterns of various genes during the induction of epigenetic rearrangements. Rat cortical neuron cultures were incubated with one of the nonselective histone deacetylase inhibitors (trichostatin A, TSA; sodium butyrate, NaB) to change the level of epigenetic regulation. Next, the total RNA was extracted and subjected to RNA-Seq libraries preparation and subsequent NGS-sequencing. Bioinformatics analysis of transcriptomic data revealed substantial overlapping of differentially expressed genes (DEGs) in NaB-treated and TSA-treated groups, indicating that different histone deacetylase (HDAC) inhibitors induce transcriptional changes in primary neuron cultures through common regulatory pathways irrespective of chemical structure of applied inhibitor. We found that histone deacetylase blockade is accompanied by a transition from proliferative processes to cellular differentiation. Gene Ontology (GO) analysis of DEGs datasets revealed that the upregulated genes were engaged in cell differentiation and specialization, tissue and embryonic morphogenesis, and the development of various peripheral tissues and organs. On the contrary, genes that reduce the expression under induction of epigenetic rearrangements were involved in biological processes associated with cell proliferation and, most interestingly, the specialization of various brain cells (neurons, astrocytes, oligodendrocytes). It was shown that the expression of a number of glial markers typical for astrocytes and oligodendrocytes was significantly reduced after application of HDAC inhibitors, which was also confirmed by quantitative PCR using specific primer pairs on selected target genes. During the data analysis, we also found a significant decrease in the expression of various neuronal markers associated with the cytoskeleton, the organization of pre- and postsynaptic endings, synaptic transmission. It is known that fine-tuning of various processes in the central nervous system is due to the production of different isoforms of proteins from the same gene due to the process of alternative splicing of the resulting mRNA. According to the literature, epigenetic rearrangements create a certain environment for the regulation of alternative splicing of different genes [2, 3]. It is shown that production of alternative isoforms can play an important role in various plastic processes [4, 5]. Therefore, using IsoformSwitchAnalyzeR and DEXSeq packages we analyzed the possibility of alternative splicing during the induction of epigenetic rearrangements in rat cortical neuron cultures by evaluating the abundance of various transcripts based on exon usage. We found that some glial genes and a large number of neuronal genes, especially those associated with postsynaptic organization and cell communication, were alternatively spliced after application of histone deacetylase inhibitors. Inhibition of HDAC activity in cortical neuron cultures mainly affected the choice of alternative transcription starts (ATSS) and terminators (ATTS), and to a lesser extent alternative splicing of exons. Obtained results were selectively confirmed by the quantitative PCR using specific pairs of primers for individual exons of different transcript isoforms. Thus, within this study, it was found that histone deacetylases play an important role in the specialization of various brain cells, and the suppression of their activity affects the expression and alternative splicing of various glial and neuronal marker genes. We do not exclude that global transcriptome changes caused by alternative splicing will lead to qualitative rearrangements of the neuron network, and this is the direction of future research.

Antidepressant Effect of Sodium Butyrate is Accompanied by Brain Epigenetic Modulation in Rats Subjected to Early or Late Life Stress.

Major depression has a complex and multifactorial etiology constituted by the interaction between genetic and environmental factors in its development. The aim of this study was to evaluate the effects of sodium butyrate (SD) on epigenetic enzyme alterations in rats subjected to animal models of depression induced by maternal deprivation (MD) or chronic mild stress (CMS). To induce MD, male Wistar rats were deprived of maternal care during the first 10 days of life. To induce CMS, rats were subjected to the CMS for 40 days. Adult rats were then treated with daily injections of SD for 7 days. Animals were subjected to the forced swimming test (FST), and then, histone deacetylase (HDAC), histone acetyltransferase (HAT), and DNA methyltransferase (DNMT) activities were evaluated in the brain. MD and CMS increased immobility time in FST and increased HDAC and DNMT activity in the animal brains. SD reversed increased immobility induced by both animal models and the alterations in HDAC and DNMT activities. There was a positive correlation between enzyme activities and immobility time for both models. HDAC and DNMT activities also presented a positive correlation between themselves. These results suggest that epigenetics can play an important role in major depression pathophysiology triggered by early or late life stress and its treatment.

HDAC6 preserves BNIP3 expression and mitochondrial integrity by deacetylating p53 at lysine 320

HDAC6 has been reported as a deacetylase of p53 at multiple lysine residues, associated with the canonical functions of p53, such as apoptosis and tumor suppression. We have previously reported that p53 acetylation at the lysine 320 site accumulates due to the genetic ablation of HDAC6 in mice liver. However, the biological processes affected by K320 acetylation of p53 are yet to be elucidated. In this study, we demonstrate that K320 acetylation of p53 is regulated by HDAC6 deacetylase activity. HDAC6 knockout mouse brains exhibit a significant accumulation of K320 acetylated p53 compared to other tissues. The level of K320 acetylation of p53 inversely correlates with the level of BNIP3, a direct target of p53 and essential for mitophagy. Notably, overexpressing the deacetylation mimic K320R mutant p53 restored BNIP3 expression in HDAC6 knockout MEFs. Furthermore, we observed that neurons are particularly susceptible to the genetic ablation of HDAC6, impacting BNIP3 expression, which inversely correlates with the accumulation of abnormal mitochondria characterized by swollen cristae. Our findings suggest that HDAC6 plays a crucial role in maintaining BNIP3 expression by deacetylating p53 at the K320 site, which is linked to the structural integrity of mitochondria.

Conserved serine phosphorylation regulates histone deacetylase activity in Arabidopsis and humans.

Conserved serine phosphorylation regulates histone deacetylase activity in Arabidopsis and humans

Effects of Dimerization on the Deacylase Activities of Human SIRT2.

Human sirtuin isoform 2 (SIRT2) is an NAD+-dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking. Dimerized SIRT2 dissociates into monomers upon binding long fatty acylated substrates (decanoyl-, dodecanoyl-, and myristoyl-lysine). However, we did not observe dissociation of dimeric SIRT2 in the presence of acetyl-lysine. Analysis of X-ray crystal structures led us to discover a SIRT2 double mutant (Q142A/E340A) that is impaired in its ability to dimerize, which was confirmed with chemical cross-linking and in cells with a split-GFP approach. In enzyme assays, the SIRT2(Q142A/E340A) mutant had normal defatty-acylase activity and impaired deacetylase activity compared with the wild-type protein. These results indicate that dimerization is essential for optimal SIRT2 function as a deacetylase. Moreover, we show that SIRT2 dimers can be dissociated by a deacetylase and defatty-acylase inhibitor, ascorbyl palmitate. Our finding that its oligomeric state can affect the acyl substrate selectivity of SIRT2 is a novel mode of activity regulation by the enzyme that can be altered genetically or pharmacologically.

Sirtuins: The NAD+-Dependent Multifaceted Modulators of Inflammation.

Significance: Sirtuins are NAD+-dependent histone deacetylases regulating important processes in cellular biology such as inflammation, metabolism, oxidative stress, and apoptosis. Recent Advances: Despite initially being discovered to regulate transcription and life span via histone deacetylase activities, emerging data continually uncover new targets and propose additional roles. Due to the outstanding importance of the sirtuins in the control of the inflammatory response, their roles in the pathogenesis of several inflammatory-based diseases have become an area of intense research. Although sirtuins have been traditionally regarded as anti-inflammatory players, several recent findings suggest that their role in inflammation is complex and that in some cases sirtuins can indeed promote inflammation. Critical Issues: In this article, we provide an update on the latest findings concerning the new mechanisms of action and concepts about the role of sirtuins during inflammation. We focus on the impact that inflammatory-based processes exert on the liver, adipose tissue, and nervous system as well as on macrophage function and activation. Also, we discuss available data pointing to the dual role that, in particular contexts, sirtuins may have on inflammation control. Future Directions: Since the knowledge of sirtuin impact on metabolism is continually expanding, new venues of research arise. Besides become being regarded as candidates of therapeutic targets, posttranscriptional control of sirtuin expression by means of microRNAs challenges our traditional concepts of sirtuin regulation; importantly, the emerging role of NAD+ metabolism in aging and longevity has added a new dimension to the interest in sirtuin function. Antioxid. Redox Signal. 39, 1185-1208.

Abstract 18596: Sildenafil Modulates Metabolic Remodeling by Increasing Fatty Acid β-Oxidation in Single Ventricle Congenital Heart Disease

Introduction: Sildenafil, a phosphodiesterase-5 inhibitor (PDE5i), is used to treat heart and circulatory failure (HF) in single ventricle congenital heart disease (SV). We previously demonstrated that human SVHF myocardium is characterized by increased PDE5 activity and impaired mitochondrial energetics, which are improved with PDE5i. Using an in vitro model in combination with SV myocardial tissue, we test the hypothesis that mitochondrial fatty acid β-oxidation (FAO) is improved with PDE5i therapy via activation of sirtuin-3 (Sirt3) and resulting protein deacetylation. Methods: Primary cardiomyocytes (neonatal rat ventricular myocytes, NRVMs) were treated with 2% serum from SVHF patients or non-failing (NF) controls +/-PDE5i (72 hours). To mechanistically interrogate whether PDE5i acts via Sirt3, we utilized siRNAs against Sirt3 +/-PDE5i. FAO was directly assessed via the Seahorse Bioanalyzer (Agilent). Mitochondrial protein levels and acetylation profiles were quantified via Western blot. Sirt3 activity was measured in isolated mitochondria from SVHF myocardium using a fluorescent-based enzymatic assay. Statistical analysis was performed using a Welch ANOVA with post-hoc Dunnett’s multiple comparisons test (FAO) or a paired T test (Sirt3 activity). Results: NRVMs treated with SVHF serum have impaired mitochondrial FAO and while PDE5i therapy results in improved FAO, siRNA knockdown of Sirt3 abolishes the PDE5i protective effect (A). Mitochondria isolated from SVHF myocardium are hyperacetylated, and ex vivo PDE5i treatment enhances Sirt3 deacetylase activity (B-C), and deacetylates mitochondrial proteins. Conclusions: SVHF serum treatment of cardiomyocytes is a novel in vitro platform for mechanistic investigations of SV-related pathophysiology. These data suggest PDE5i directly augments mitochondrial FAO via Sirt3-mediated protein deacetylation and provides important insight into the myocardial specific mechanisms of PDE5i.