Removal Of Acetyl Groups Research Articles

Acetylation‐phosphorylation cross‐talk: A role for HDACs in the regulation of PKCdelta/theta phosphorylation

Post‐translational modifications (PTMs) such as acetylation and phosphorylation have been implicated in contractile dysfunction and muscle wasting. Moreover, mounting evidence demonstrates that histone deacetylase (HDAC) inhibitors attenuate muscle wasting in animal models of disuse and muscular dystrophy. Historically, lysine acetylation was studied in the regulation of gene transcription, where for instance HDACs remove acetyl groups on lysine residues of histone proteins to suppress gene expression. However, recent evidence has shown that non‐histone proteins are also regulated by acetylation; functioning in protein stability, intracellular signaling, mitochondrial function and muscle contraction. Despite our current understanding for lysine acetylation and protein phosphorylation in muscle, how these PTMs cross‐talk to regulate muscle physiology remains unclear. In our attempt to understand acetylation‐phosphorylation cross‐talk, we examined the role for HDAC inhibitors on protein kinase C delta (PKCδ) and delta/theta (PKCδ/θ) phosphorylation. PKC is composed of multiple isoforms and its activation has been linked to muscle disease and dysfunction. Myotubes were treated with or without a pan‐HDAC inhibitor (trichostatin A; TSA) in the absence or presence of a pathological agonist (i.e. PMA). Phorbol 12‐myristate 13‐acetate (PMA) increased phosphorylation of PKCδ and PKCδ/θ. Interestingly, PMA‐induced PKCδ and PKCδ/θ phosphorylation was amplified in myotubes treated with the pan‐HDAC inhibitor TSA. Class selective HDAC inhibitors confirmed these findings and further demonstrated that inhibition of class I HDACs (HDAC1, 2, 3) was sufficient to increase PKCδ and PKCδ/θ phosphorylation. It should be noted that total PKCδ and PKCδ/θ protein expression increased with class I HDAC inhibition concomitant to the increase in protein phosphorylation. These data suggest that HDAC inhibition increases total PKC gene/protein expression that can then be phosphorylated. Finally, we report that the HDAC 1/2 selective inhibitor Romidepsin also increased total and phosphorylated PKCδ and PKCδ/θ in response to PMA. Genetic knockdown studies are currently underway to elucidate the specific HDAC that regulates PKC gene/protein expression, while chromatin immunoprecipitation (ChIP) is being used to determine HDAC binding to the PKC promoter. Lastly, elucidating a functional consequence for HDAC inhibitor‐mediated regulation of PKCδ and PKCδ/θ protein expression and phosphorylation in muscle could yield significant insight into muscle wasting and function.Support or Funding InformationThis work is supported by the USDA NIFA (Hatch‐NEV00727, Hatch‐NEV00767), the Dennis Meiss & Janet Ralston Fund for Nutri‐epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459) and the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496) and NSF EPSCOR Track II (OIA‐1826801) to B.S.F.

Exploring the inhibitory activity of valproic acid against the HDAC family using an MMGBSA approach.

Valproic acid (VPA) is a compound currently used in clinical practice for the treatment of epilepsy as well as bipolar and mood disorders. VPA targets histone deacetylases (HDACs), which participate in the removal of acetyl groups from lysine in several proteins, regulating a wide variety of functions within the organism. An imbalance or malfunction of these enzymes is associated with the development and progression of several diseases, such as cancer and neurodegenerative diseases. HDACs are divided into four classes, but VPA only targets Class I (HDAC1-3 and 8) and Class IIa (HDAC4-5, 7 and 9) HDACs; however, structural and energetic information regarding the manner by which VPA inhibits these HDACs is lacking. Here, the structural and energetic features that determine this recognition were studied using molecular docking and molecular dynamics (MD) simulation. It was found that VPA reaches the catalytic site in HDAC1-3 and 7, whereas in HDAC6, VPA only reaches the catalytic tunnel. In HDAC4, VPA was bound adjacent to L1 and L2, a zone that participates in corepressor binding, and in HDAC8, VPA was bound to the hydrophobic active site channel (HASC), in line with previous reports.

Emerging Concepts on the Role of ADP-Ribosylation

NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways, since it acts as a key regulator of cellular and organism homeostasis. NAD+ is a coenzyme in redox reactions, a donor of adenosine diphosphate-ribose (ADPr) moieties in ADP-ribosylation reactions, and a substrate for sirtuins, a group of histone deacetylase enzymes that use NAD+ to remove acetyl groups from proteins. NAD+ is also a precursor of cyclic ADP-ribose, a second messenger in the release and signaling of Ca++, and of diadenosine tetraphosphate (Ap4A) and oligoadenylates (oligo2′-5′A)—two immune response-activating compounds. In the biological systems considered in this review, NAD+ is mostly consumed in ADP-ribose (ADPr) transfer reactions. In this review, the roles of these chemical products are discussed in biological systems, such as in animals, plants, fungi and bacteria. In the review, ADP-ribosylating enzymes are introduced, as well as the importance to restore the NAD+ pools in these systems. Finally, a special attention is presently focused on viral macrodomains, aimed to develop inhibitors to improve the immune response to viruses.

The Role of Histone Acetyltransferases and Histone Deacetylases in Photoreceptor Differentiation and Degeneration.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electrical signals. The differentiation and degeneration of photoreceptors are regulated by specific genes and proteins. With the development of epigenetic approaches, scientists have discovered that histone modifications, such as acetylation, methylation, ubiquitylation, and phosphorylation, may modulate the processes of photoreceptor differentiation and degeneration. Histone acetylation is regulated by two opposing classes of enzymes, namely, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which add and remove acetyl groups to and from target histones, respectively, causing changes in transcriptional activity. Herein, we review the effects of HATs and HDACs on the differentiation and degeneration of photoreceptors and discuss the underlying mechanisms of these effects.

Functions and mechanisms of plant histone deacetylases.

Lysine acetylation, one of the major types of post-translational modifications, plays critical roles in regulating gene expression and protein function. Histone deacetylases (HDACs) are responsible for removing acetyl groups from lysines of both histone and non-histone proteins. While tremendous progress has been made in understanding the function and mechanism of HDACs in animals in the past two decades, nearly half of the HDAC studies in plants were reported within the past five years. In this review, we summarize the major findings on plant HDACs, with a focus on the model plant Arabidopsis thaliana, and highlight the components, regulatory mechanisms, and biological functions of HDAC complexes.

Trichostatin A inhibits skeletal muscle atrophy induced by cigarette smoke exposure in mice

AimsIt is well known that cigarette smoke (CS) is the main risk factor for chronic obstructive pulmonary disease (COPD) accompanied by skeletal muscle atrophy. Histone deacetylases (HDACs) that remove acetyl groups from target proteins are necessary for the muscle atrophy associated with skeletal muscle disuse. However, the role of HDACs and trichostatin A (TSA), a HDAC inhibitor, in skeletal muscle atrophy caused by CS exposure remains poorly understood. Main methodsFemale mice were exposed to CS twice daily for 40 days and TSA injected intraperitoneally into CS-exposed mice on alternate days. Skeletal muscles were weighed and gastrocnemius (Gas) muscle histomorphology examined by hematoxylin and eosin staining. Histone deacetylases 1 and 2 (HDAC1/2), and markers of ubiquitin degradation, muscle differentiation, apoptosis, pyroptosis, and the cytoskeletal proteins were assessed by western blot and immunohistochemistry in Gas. KeyfindingsCS exposure decreased body and skeletal muscle weights and triggered an increase in the percentage of fiber with centralized nuclei in Gas. HDAC1/2 proteins were upregulated in the Gas of mice exposed to CS, while TSA effectively inhibited HDAC1/2 protein levels and attenuated the loss of body weight and skeletal muscle wet weight induced by CS exposure. Markers for ubiquitin degradation, muscle differentiation, cytoskeletal proteins, apoptosis and pyroptosis were all upregulated following CS exposure and effectively restored by TSA. SignificanceTSA may inhibit skeletal muscle atrophy and histomorphological alterations induced by CS exposure by downregulating markers of ubiquitin degradation, muscle fiber differentiation, cytoskeletal proteins, apoptosis and pyroptosis via HDAC1/2 inhibition.

Knockout of longevity gene Sirt1 in zebrafish leads to oxidative injury, chronic inflammation, and reduced life span.

Sirt1, a member of the sirtuin gene family, encodes the most conserved mammalian NAD+-dependent deacetylase enzyme responsible for removing acetyl groups from many proteins. The Sirt1 gene is known as a longevity gene whose knockout in mice leads to decreased lifespan relative to the wild type. This study aimed to explore phenotypic changes in zebrafish Sirt1-knockouts and to investigate the function of the Sirt1 gene. Targeted knockout of Sirt1 in zebrafish (Danio rerio) was achieved using the CRISPR-Cas9 genome editing system. We created a 4-bp insertion-homozygote Sirt1-knockout zebrafish. Although there was no evident difference in appearance in the early stages of development, a significant increase in reactive oxygen species and in the extent of apoptosis in Sirt1-knockout zebrafish was observed. Sirt1 knockout caused inflammatory genes, including IL-1b, IL-6 and TNF-α to be highly expressed. Additionally, the lack of Sirt1 caused chronic inflammation and intestinal atrophy, thereby increasing pro-apoptotic events, which ultimately reduced the lifespan of transgenic zebrafish. Overall, our data demonstrate that lack of Sirt1 caused a significantly increased generation of reactive oxygen species that resulted in chronic inflammation and regeneration. Continuous repetition of these events played an important role in accelerating aging, thereby decreasing lifespan. Our findings using the knockout zebrafish model confirmed the association of the Sirt1 gene to aging processes and lifespan. Furthermore, the Sirt1-knockout mutant zebrafish developed in our study will surely be a valuable model to explore the mechanism of chronic inflammation.

CURCUMIN AS A NOVEL EPIGENETIC TREATMENT APPROACH FOR GFI1-ASSOCIATED MDS/AML

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are diseases caused by an ineffective myelopoiesis. AML is an aggressive blood cancer with poor prognosis for patients despite treatment with intensive chemotherapy, hence alternative therapies are needed. MDS and AML patients with low expression of the transcriptional repressor GFI1 (Growth factor independence 1) or expression of the GFI1 variant GFI1-36N (asparagine instead of serine at amino acid position 36) in their blast cells have an even poorer prognosis. GFI1 recruits amongst others HDAC1 and 2 (histone deacetylase 1 and 2) to its target genes. On a molecular level, expression of GFI1 thus leads to the removal of acetyl groups at H3K9. Low GFI1 (GFI1-KD) or GFI1-36N expression in MDS and AML blasts resulted in increased H3K9ac at its target genes, causing elevated target gene expression. Some of these target genes were oncogenes, explaining why GFI1-KD and GFI1-36N promote AML development. We hypothesized that administration of HAT (histone acetyltransferase) inhibitors could be beneficial for MDS/AML patients with reduced GFI1 or GFI1-36N expression as it could reverse the increased acetylation of H3K9. Curcumin is a HAT inhibitor which is used as a spice with so far no known toxic side effects. To study the effect of Curcumin on MDS/AML development, we crossed the well-established murine model of human MDS/AML, NUP98-HOXD13, with GFI1-WT, GFI1-KD or GFI1-36N mice. The different groups were treated with either curcumin or were left untreated. Curcumin effectively prevented the development of AML in mice with low GFI1 or GFI1-36N expression, but not in GFI1-WT mice. Overall, our data suggest that GFI1 functions as a prognostic marker in MDS/AML patients, resulting in a personalized treatment approach using Curcumin in patients with low GFI1 or GFI1-36N expression.

Development of Multifunctional Histone Deacetylase 6 Degraders with Potent Antimyeloma Activity.

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as α-tubulin and HSP90. HDAC6 is involved in multiple disease-relevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC50 and promising antiproliferation activity in multiple myeloma (MM) cells.

RENDEMEN KITOSAN LIMBAH CANGKANG KERANG SIMPING (Amusium pleuronectes) DAN KERANG KEPAH (Polymesoda erosa) DARI KENDAL JAWA TENGAH

ABSTRACTChitosan is the result of the deacetylation process of chitin by removing acetyl groups to obtain polymer compounds and glucosamine. Utilization of chitosan derived from waste of Amusium pleuronectes and Polymesoda erosa has not been maximize. This study aims to determine the highest chitosan yield in Amusium pleuronectes and Polymesoda erosa. The method used in this study was a deacetylation isolation method with five replications. The yield of the yield content was analyzed by univariate and bivariate with spps 19.0 using a ttest. Based on the results of the research on the sample of Amusium pleuronectes and Polymesoda erosa, the highest yield of chitosan at 75% b/v concentration of NaOH was 6,972% b/v for Amusium pleuronectes and 5.972% b/v for Polymesoda erosa. The results showed that NaOH concentrations of 15v, 25, 50, and 75% b/v could affect the chitin change to chitosan against Amusium pleuronectes and Polymesoda erosa.Keywords: Amusium pleuronectes, Polymesoda erosa, deacetylation, chitin, chitosan

Regulation of HDAC1 and HDAC2 during consolidation and extinction of fear memory

Histone deacetylases (HDACs) regulate gene expression epigenetically through synchronized removal of acetyl groups from histones required towards memory consolidation. Moreover, dysregulated epigenetic machinery during fear or extinction learning may result in altered expression of some of these genes and result in Post Traumatic Stress Disorder (PTSD). In the present study, region-specific expression of Histone deacetylase 1 (HDAC1) and Histone deacetylase 2 (HDAC2) was correlated to the acetylation of histones H3 and H4 and the resultant conditioned response, in rats undergone fear and extinction learning. The neuronal activation, histone acetylation at H3/H4 and expression of HDAC1/HDAC2 in centrolateral amygdala (CeL) and centromedial amygdala (CeM) of central Amygdala (CeA) and prelimbic (PL) and infralimbic (IL) of Prefrontal cortex (PFC) were found to be associated in a differential manner following fear and extinction learning. Moreover in CeM, the main output of the fear circuitry, the level of HDAC1 was down-regulated following conditioning and up-regulated following extinction as opposed to which HDAC2 was down-regulated in CeM following conditioning but not following extinction. Furthermore, in CeL the HDAC1 was upregulated and HDAC2 was downregulated following conditioning and extinction. This has important implications in speculating of the role of HDACs in fear memory consolidation and its extinction.

MYB96 recruits the HDA15 protein to suppress negative regulators of ABA signaling in Arabidopsis

Unlike activation of target genes in response to abscisic acid (ABA), how MYB96 transcription factor represses ABA-repressible genes to further enhance ABA responses remains unknown. Here, we show MYB96 interacts with the histone modifier HDA15 to suppress negative regulators of early ABA signaling. The MYB96-HDA15 complex co-binds to the promoters of a subset of RHO GTPASE OF PLANTS (ROP) genes, ROP6, ROP10, and ROP11, and represses their expression by removing acetyl groups of histone H3 and H4 from the cognate regions, particularly in the presence of ABA. In support, HDA15-deficient mutants display reduced ABA sensitivity and are susceptible to drought stress with derepression of the ROP genes, as observed in the myb96-1 mutant. Biochemical and genetic analyses show that MYB96 and HDA15 are interdependent in the regulation of ROP suppression. Thus, MYB96 confers maximal ABA sensitivity by regulating both positive and negative regulators of ABA signaling through distinctive molecular mechanisms.

Physico-chemical and functional properties of silver carp myosin glycated with konjac oligo-glucomannan: Effects of deacetylation

The glycation of silver carp myosin (Ms) with konjac oligo-glucomannan (KOG) of different degrees of deacetylation (DD) was investigated. As DD increased, the physico-chemical and functional properties of glycoconjugates changed. The available lysine content decreased, while grafting degree and total sulfhydryl group increased, andmeanwhile, the isoelectric point (pI) reduced. SDS-PAGE analysis indicated that glycation can be promoted as the increase of DD. The solubility increased significantly both in 0.1 M and 0.5 M NaCl solution, and the thermal stability increased when heated for 60 min at 80 °C. The emulsifying activity index (EAI) and emulsifying stability index (ESI) increased as well. These results showed that the highly deacetylated KOG (DKOG) was easier to glycate with myosin, leading to a great improvement in functional properties of myosin. It can be suggested that the reduction in steric hindrance of DKOG as a result of removal of acetyl groups facilitated the glycation.

Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family

Insect chitin deacetylases (CDAs) catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. However, the structural and biochemical characteristics of insect CDAs have remained elusive. Here, we report the crystal structures of two insect CDAs from the silk moth Bombyx mori: BmCDA1, which may function in cuticle modification, and BmCDA8, which may act in modifying peritrophic membranes in the midgut. Both enzymes belong to the carbohydrate esterase 4 (CE4) family. Comparing their overall structures at 1.98–2.4 Å resolution with those from well-studied microbial CDAs, we found that two unique loop regions in BmCDA1 and BmCDA8 contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both BmCDA1 and BmCDA8 possess a much longer and wider substrate-binding cleft with a very open active site in the center than the microbial CDAs, including VcCDA from Vibrio cholerae and ArCE4A from Arthrobacter species AW19M34-1. Biochemical analyses indicated that BmCDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, BmCDA1 also required accessory proteins for catalysis. To the best of our knowledge, our work is the first to unveil the structural and biochemical features of insect proteins belonging to the CE4 family.

Low levels of pyruvate induced by a positive feedback loop protects cholangiocarcinoma cells from apoptosis

BackgroundCancer cells avidly consume glucose and convert it to lactate, resulting in a low pyruvate level. This phenomenon is known as the Warburg effect, and is important for cell proliferation. Although cMyc has often been described as an oncoprotein that preferentially contributes to the Warburg effect and tumor proliferation, mechanisms of action remain unclear. Histone deacetylase 3 (HDAC3) regulates gene expression by removing acetyl groups from lysine residues, as well as has an oncogenic role in apoptosis and contributes to the proliferation of many cancer cells including cholangiocarcinoma (CCA). HDAC inhibitors display antitumor activity in many cancer cell lines. Cancer cells maintain low levels of pyruvate to prevent inhibition of HDAC but the mechanisms remain elusive. The purpose of our study was to explore the role of cMyc in regulating pyruvate metabolism, as well as to investigate whether the inhibitory effect of pyruvate on HDAC3 could hold promise in the treatment of cancer cells.MethodsWe studied pyruvate levels in CCA cell lines using metabolite analysis, and analyzed the relationship of pyruvate levels and cell proliferation with cell viability analysis. We cultivated CCA cell lines with high or low levels of pyruvate, and then analyzed the protein levels of HDAC3 and apoptotic markers via Western Blotting. We then explored the reasons of low levels of pyruvate by using seahorse analysis and 13C6 metabolites tracing analysis, and then confirmed the results using patient tissue protein samples through Western Blotting. Bioinformatics analysis and transfection assay were used to confirm the upstream target of the low levels of pyruvate status in CCA. The regulation of cMyc by HDAC3 was studied through immunoprecipitation and Western Blotting.ResultsWe confirmed downregulated pyruvate levels in CCA, and defined that high pyruvate levels correlated with reduced cell proliferation levels. Downregulated pyruvate levels decreased the inhibition to HDAC3 and consequently protected CCA cells from apoptosis. Synergistically upregulated LDHA, PKM2 levels resulted in low levels of pyruvate, as well as poor patient survival. We also found that low levels of pyruvate contributed to proliferation of CCA cells and confirmed that the upstream target is cMyc. Conversely, high activity of HDAC3 stabilized cMyc protein by preferential deacetylating cMyc at K323 site, which further contributed to the low pyruvate levels. Finally, this creates a positive feedback loop that maintained the low levels of pyruvate and promoted CCA proliferation.ConclusionsCollectively, our findings identify a role for promoting the low pyruvate levels regulated by c-Myc, and its dynamic acetylation in cancer cell proliferation. These targets, as markers for predicting tumor proliferation in patients undergoing clinical treatments, could pave the way towards personalized therapies.

Inhibition of BET Proteins and Histone Deacetylase (HDACs): Crossing Roads in Cancer Therapy.

Histone DeACetylases (HDACs) are enzymes that remove acetyl groups from histones and other proteins, regulating the expression of target genes. Pharmacological inhibition of these enzymes re-shapes chromatin acetylation status, confusing boundaries between transcriptionally active and quiescent chromatin. This results in reinducing expression of silent genes while repressing highly transcribed genes. Bromodomain and Extraterminal domain (BET) proteins are readers of acetylated chromatin status and accumulate on transcriptionally active regulatory elements where they serve as scaffold for the building of transcription-promoting complexes. The expression of many well-known oncogenes relies on BET proteins function, indicating BET inhibition as a strategy to counteract their activity. BETi and HDACi share many common targets and affect similar cellular processes to the point that combined inhibition of both these classes of proteins is regarded as a strategy to improve the effectiveness of these drugs in cancer. In this work, we aim to discuss the molecular basis of the interplay between HDAC and BET proteins, pointing at chromatin acetylation as a crucial node of their functional interaction. We will also describe the state of the art of their dual inhibition in cancer therapy. Finally, starting from their mechanism of action we will provide a speculative perspective on how these drugs may be employed in combination with standard therapies to improve effectiveness and/or overcome resistance.

HDA9-PWR-HOS15 Is a Core Histone Deacetylase Complex Regulating Transcription and Development.

Histone deacetylases remove acetyl groups from histone proteins and play important roles in many genomic processes. How histone deacetylases perform specialized molecular and biological functions in plants is poorly understood. Here, we identify HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15) as a core member of the Arabidopsis (Arabidopsis thaliana) HISTONE DEACETYLASE9-POWERDRESS (HDA9-PWR) complex. HOS15 immunoprecipitates with both HDA9 and PWR. Mutation of HOS15 induces histone hyperacetylation and methylation changes similar to hda9 and pwr mutants. HOS15, HDA9, and PWR are coexpressed in all organs, and mutant combinations display remarkable phenotypic resemblance and nonadditivity for organogenesis and developmental phase transitions. Ninety percent of HOS15-regulated genes are also controlled by HDA9 and PWR HDA9 binds to and directly represses 92 genes, many of which are responsive to biotic and abiotic stimuli, including a family of ethylene response factor genes. Additionally, HOS15 regulates HDA9 nuclear accumulation and chromatin association. Collectively, this study establishes that HOS15 forms a core complex with HDA9 and PWR to control gene expression and plant development.

Modification of corn stover for improving biodegradability and anaerobic digestion performance by Ceriporiopsis subvermispora

Ceriporiopsis subvermispora was used to modify corn stover for improving the biodegradability and biomethane yield. Corn stover was incubated with C. subvermispora for 5–90 days then anaerobically digested. It was found that the corn stover modified for 15 days achieved the highest biomethane yield of 235 mL·g−1 VS, which was an increase of 15.2% over that of the non-modified one. The mechanism analyses indicated that the improvement resulted from the combined roles of degradation selectivity, destruction of lignocellulosic structures, and linkages. The analyses showed that C. subvermispora has a high relative selectivity of lignin degradation. The structure of the lignin and the linkages among lignin and hemicellulose and cellulose were broken obviously by acetyl group removal, and the enzymatic hydrolysis of cellulose was increased by 35.61%. The finding indicated that C. subvermispora modification is one of the effective methods for enhancing biomethane yield of corn stover.

Acetylation of Actin K328 Contributes to a Loss in Tropomyosin-Mediated Inhibition of Myosin Binding

Lysine deacetylases (KDAC's) catalyze the removal of acetyl groups from lysine side chains, while lysine acetylases (KAT's) perform the reverse reaction. KDAC inhibition was shown to increase lysine acetylation of muscle proteins, and was associated with enhanced Ca2+-sensitivity in skinned fibers and increased relaxation rate in isolated myofibrils. Interestingly, the KAT enzyme, PCAF, directly acetylates myosin in vitro, and actin has been implicated as a potential target of PCAF. Therefore, we attempted to acetylate actin with PCAF, in vitro, to determine its potential effect(s) on muscle function. PCAF underwent autoacetylation and bound to actin, yet failed to acetylate it. Chemical treatment of actin with acetic anhydride, however, increased lysine acetylation roughly 200-fold. Surprisingly, in vitro motility (IVM) of (100 μg/ml myosin concentration) and tropomyosin affinity for acetylated F-actin were indistinguishable from unacetylated control. However, IVM of acetylated F-actin, performed at low myosin concentration (12.5 μg/ml) and in the presence of tropomyosin, revealed a significant 63% increase in motile filaments relative to unacetylated control (p < 0.0001), suggesting that actin acetylation reduces tropomyosin-mediated inhibition of myosin binding. Since K326 and K328 have been reported as the most reactive lysines on actin, are acetylated in vivo, and pseudo-acetylation of both modulates muscle contraction, we assessed the effect of K326Q and K328Q pseudo-acetylated actin on Ca2+-mediated thin filament activation via regulated IVM. Although reconstituted thin filaments containing K326Q (pCa50 = 6.36 ± 0.03) did not alter Ca2+-sensitivity relative to WT (pCa50 = 6.37 ± 0.03), K328Q induced a significant leftward shift in Ca2+-sensitivity (ΔpCa50 = 0.19; p < 0.0001), consistent with a loss in tropomyosin-mediated inhibition. The data indicate that actin K328 acetylation modulates contractile function, which makes it a potential mechanism for regulating muscle performance in vivo.

Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells

BackgroundThe diagnosis of glioblastoma (GBM), a most aggressive primary brain tumor with a median survival of 14.6 months, carries a dismal prognosis. GBMs are characterized by numerous genetic and epigenetic alterations, affecting patient survival and treatment response. Epigenetic mechanisms are deregulated in GBM as a result of aberrant expression/activity of epigenetic enzymes, including histone deacetylases (HDAC) which remove acetyl groups from histones regulating chromatin accessibility. Nevertheless, the impact of class/isoform-selective HDAC inhibitors (HDACi) on glioma cells, including glioma stem cells, had not been systematically determined.ResultsComprehensive analysis of the public TCGA dataset revealed the increased expression of HDAC 1, 2, 3, and 7 in malignant gliomas. Knockdown of HDAC 1 and 2 in human GBM cells significantly decreased cell proliferation. We tested the activity of 2 new and 3 previously described HDACi with different class/isoform selectivity on human GBM cells. All tested compounds exerted antiproliferative properties on glioma cells. However, the HDACi 1 and 4 blocked proliferation of glioblastoma cells leading to G2/M growth arrest without affecting astrocyte survival. Moreover, 1 and 4 at low micromolar concentrations displayed cytotoxic and antiproliferative effects on sphere cultures enriched in glioma stem cells.ConclusionsWe identified two selective HDAC inhibitors that blocked proliferation of glioblastoma cells, but did not affect astrocyte survival. These new and highly effective inhibitors should be considered as promising candidates for further investigation in preclinical GBM models.